Inhibitors of microbially induced amyloid

ABSTRACT

The present disclosure provides compounds useful for the prevention of amyloid formation and the treatment of amyloid related disorders, including synucleopathies such as Parkinson&#39;s Disease.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(c) to U.S.provisional patent application. U.S. Ser. No. 62/541,536, filed Aug. 4,2017, the entire contents of which is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to inhibitors of amyloid formation, andparticularly inhibitors of microbially-induced amyloid formation, aswell as methods of using such inhibitors to treat or inhibitneurological disorders and other disorders associated with amyloidaccumulation. Methods of identifying compositions that inhibit orpromote amyloid formation are also provided.

BACKGROUND

Many neurodegenerative diseases are associated with atypical aggregationof proteins in the brain, which leads to cell death and a resultingmanifestation of many neuropathies. It is believed that diseasespecificity is a consequence of (i) the specific proteins involved inaggregation. (ii) the specific regions of the brain affected, and (iii)the specific neuronal cell types affected. In the case of the naturalhuman protein α-synuclein, aberrant aggregation of this protein leads toany of over 50 “α-synucleinopathies,” of which Parkinson's Disease isthe most common and most widely studied. In Parkinson's Disease.α-synuclein aggregation leads to the accumulation of large precipitatedaggregates, called Lewy bodies, within certain neuronal cell types, mosttypically those that produce the neurotransmitter dopamine. When enoughα-synuclein aggregate is present, neuronal death occurs and dopamineproduction declines. Dopamine is required for proper control ofmovement, and once dopaminergic neurons are killed they are notreplaced. Over time the dopamine pool declines irreversibly to a pointwhere motor symptoms progress and become debilitating.

The most pathogenic form of α-synuclein is still unclear, e.g., whetherfull intact Lewy bodies or smaller oligomeric α-synuclein fibrils aremost relevant to disease progression and pathology. Traditionalpharmaceutical and biotech approaches to inhibiting α-synucleinaggregation have focused on attacking the aggregation process in theneurons and brain regions most associated with disease symptoms. Smallmolecule approaches, antibody approaches and a vaccine approach all havebeen attempted and continue to be evaluated as interventions forParkinson's Disease and other α-synucleinopathies. Importantly, all ofthese strategies presently rely on the therapeutic entity crossing theblood-brain barrier and reaching the target neuronal tissue. Traversingthe blood-brain barrier remains one of the most significantpharmacokinetic challenges that hinders drug development forneurodegenerative diseases. Accordingly, there is a need for inhibitorsof amyloid formation, and especially α-synuclein aggregation inhibitors,that have the potential for providing therapeutic effects without havingto cross the blood-brain barrier.

SUMMARY

In some embodiments, the composition is formulated for delivery outsideof the systemic circulation of a subject. Said composition may beformulated for enteric or intranasal delivery, for example, and/or saidcompositions may further be formulated for controlled release within thelower intestine or colon. The aforementioned compositions may comprisean enteric-coated capsule, tablet, soft-gel, spray dried powder, polymermatrix, hydrogel, enteric-coated solid, crystalline solid, amorphoussolid, glassy solid, coated micronized particle, liquid, nebulizedliquid, aerosol, or microcapsule.

The present disclosure further provides a method of disrupting theformation of amyloid aggregates, comprising contacting an amyloid or aprecursor of an amyloid with a composition comprising one or morecompounds of the invention.

The present disclosure further provides a method of disrupting theformation of amyloid aggregates in a subject, comprising administeringto said subject a composition comprising one or more polyphenols orpolyphenol-like compounds, such as a composition comprising a compoundof the invention. Optionally, said subject is additionally selected oridentified as one that would receive the benefit of a molecule thatdisrupts the formation of amyloid aggregates prior to administration ofsaid composition. Such selection or identification can be made byclinical or diagnostic evaluation, prior to administering saidcomposition. Such selected subjects may have been diagnosed or evaluatedfor Parkinson's Disease. Lewy Body Dementia, incidental Lewy bodydisease, Lewy body variant of Alzheimer's disease, multiple systematrophy, or pure autonomic failure, or any combination thereof.Optionally, prior to, during or after administration of the composition,the disruption or inhibition of the formation of amyloid aggregates insaid subject is measured or evaluated.

The present disclosure further provides a method of inhibiting,ameliorating, reducing the likelihood, delaying the onset of, treating,or preventing an amyloid disorder in a subject. The method can compriseadministering to the subject a composition comprising a compound of theinvention, as described herein. Optionally, the subject is additionallyselected or identified as one that would receive the benefit of amolecule that inhibits or disrupts the formation of amyloid aggregatesprior to administration of said composition, for example by detecting apresence or level of a bacterial protein (such as CsgA), or a presenceor level of a microbial organism that makes the bacterial protein in anintestinal sample of the subject. Such selection or identification canbe made by clinical or diagnostic evaluation, prior to administeringsaid composition. Such selected subjects may have been diagnosed orevaluated for Parkinson's Disease. Lewy Body Dementia, incidental Lewybody disease. Lewy body variant of Alzheimer's disease, multiple systematrophy, or pure autonomic failure, or any combination thereof.Optionally, prior to, during or after administration of the composition,the disruption or inhibition of the formation of amyloid aggregates insaid subject is measured or evaluated.

In some embodiments according to the methods and compositions asdescribed herein, said amyloid aggregates may comprise one or moremammalian amyloid or mammalian amyloid precursors such as proteins,and/or one or more bacterial or fungal proteins (e.g., a compositioncomprising CsgA). In some embodiments according to the methods andcompositions disclosed herein, said amyloid aggregates may be presentwithin the gastrointestinal tract, the enteric nervous tissue, cranialsinus, or nasal cavity (e.g., the olfactory bulb).

In some embodiments, the methods of the present disclosure furthercomprise measuring or evaluating a change in the subject's nervoussystem, such as a neurological symptom, motor behavior, or otherbehavior of the subject, which may comprise, e.g., one or more ofanosmia, hyposmia, bradykinesia, ataxia, tremor, muscle rigidity,impaired posture and balance, loss of automatic movements, dysarthria orother speech changes, handwriting changes, orthostatic hypotension,memory deficit, dysphagia, incontinence, sleep disruption, cardiacarrhythmia, visual disturbance, psychiatric problems includingdepression and/or visual, auditory, olfactory, and/or tactilehallucinations, vertigo, cognitive dysfunction, altered dopamine levels,altered serotonin levels, altered kynurenine levels, or any combinationthereof.

In some embodiments, the methods of the present disclosure furthercomprise measuring or evaluating a change in the gastrointestinalsystem, such as a gastrointestinal symptom or behavior of the subject,which may comprise, e.g., one or more of dysphagia, reduced gutmotility, gastroparesis, constipation (including chronic constipationand chronic idiopathic constipation), small intestine bacterialovergrowth (SIBO), diarrhea, abdominal pain and/or cramping, bloating,flatulence, nausea, or any other symptoms of irritable bowel syndrome(IBS), inflammatory bowel disease (IBD), ulcerative colitis. Crohn'sdisease, intestinal hyperpermeability, leaky gut, intestinal dysbiosis,hypersalivation (sialorrhea), anorectal dysfunction, dyssynergicdefecation, or any combinations thereof. As used herein, the terms IBSand IBD have their customary and ordinary meaning as understood by oneof skill in the art in view of this disclosure. Such hyperpermeabilitymay result from inflammation of the intestinal lining and/or failure ofthe tight junctions between cells of the intestinal epithelium, whichallows the passage of substances from the lumen into the surroundingtissues where some may enter the peritoneal cavity and/or systemiccirculation. Because of this leakage of substances from the gut orintestinal lumen, intestinal hyperpermeability may be referred to as“leaky gut” or “leaky gut syndrome.”

In some embodiments, the compositions of the present disclosure may beadministered to a subject prior to, or following, the appearance of aneurological symptom or condition. In some embodiments, the compositionsof the present disclosure may be administered to a subject prior to, orfollowing, the appearance of a gastrointestinal symptom or conditionassociated with an amyloid disorder. In some embodiments, said subjectis selected as one that has been identified as being at risk fordeveloping or already having Lewy Body Dementia, incidental Lewy bodydisease, Lewy body variant of Alzheimer's disease, multiple systematrophy, pure autonomic failure, or any combination thereof, such as byclinical or diagnostic evaluation. In some embodiments, said subject isunder the age of 18, 18-30, 30-50, 50-60, 60-70, or over the age of 70.In some embodiments, said subject is one that has been identified orselected as being at risk for developing or already having Parkinson'sdisease, such as by clinical or diagnostic evaluation or family historyanalysis.

In some embodiments according to the compositions and methods disclosedherein said composition may be coadministered with caffeine, nicotine,theophylline, theobromine, xanthine, methylxanthine, or derivativesthereof. In some embodiments, the methods as disclosed herein furthercomprise administering to said subject an inhibitor of α-synucleinaggregation. In some embodiments, the methods as disclosed hereinfurther comprise administering to said subject L-DOPA, carbodopa,levodopa, Droxidopa, rasagiline, apomorphine hydrochloride,Bromocriptine, Rotigotine, Pramipexole, Ropinirole, Benzotropine,Trihexyphenidyl, Selegiline, Entacapone, Tolcapone, Amantadine,Pimavanserin, Rivastigmine or the like, or any combination thereof. Insome embodiments, the methods as disclosed herein comprise administeringto said subject an inhibitor of α-synuclein aggregation, and furthercomprise administering to said subject L-DOPA, carbodopa, levodopa,Droxidopa, rasagiline, apomorphine hydrochloride, Bromocriptine,Rotigotine, Pramipexole, Ropinirole, Benzotropine, Trihexyphenidyl,Selegiline, Entacapone, Tolcapone, Amantadine, Pimavanserin.Rivastigmine or the like, or any combination thereof. In someembodiments, the inhibitor of α-synuclein aggregation and the L-DOPA,carbodopa, levodopa, Droxidopa, rasagiline, apomorphine hydrochloride,Bromocriptine, Rotigotine, Pramipexole, Ropinirole, Benzotropine,Trihexyphenidyl, Selegiline, Entacapone, Tolcapone, Amantadine,Pimavanserin, Rivastigmine or the like are administered in the samecomposition. In some embodiments, the inhibitor of α-synucleinaggregation and the L-DOPA, carbodopa, levodopa, Droxidopa, rasagiline,apomorphine hydrochloride, Bromocriptine, Rotigotine, Pramipexole,Ropinirole, Benzotropine, Trihexyphenidyl, Selegiline, Entacapone,Tolcapone, Amantadine, Pimavanserin, Rivastigmine or the like areadministered in separate compositions. In some embodiments, the separatecompositions are administered at the same time. In some embodiments, theseparate compositions are administered at the different times.

In some embodiments, the composition according to any of thecompositions and methods disclosed herein is for medical use. In someembodiments, the composition according to any of the compositions andmethods disclosed herein is for use in treating an amyloid disorder asdescribed herein (such as an amyloid disorder of Table 3). In someembodiments, the amyloid disorder is selected from the group consistingof Parkinson's Disease, Lewy Body Dementia, incidental Lewy bodydisease. Lewy body variant of Alzheimer's disease, multiple systematrophy, or pure autonomic failure, or any combination of two or more ofthese. In some embodiments the composition according to the compositionsand methods disclosed herein is for use in preparing a medicament forthe treatment for an amyloid disorder as described herein (such as anamyloid disorder of Table 3). In some embodiments, the amyloid disorderis selected from the group consisting of Parkinson's Disease, Lewy BodyDementia, incidental Lewy body disease, Lewy body variant of Alzheimer'sdisease, multiple system atrophy, or pure autonomic failure, or anycombination of two or more of these. In some embodiments, thecomposition comprises one or more compounds of the invention, asdescribed herein. In some embodiments, the composition is formulated fordelivery to the gastrointestinal tract, for example via oral or rectaldelivery, or formulated with an enteric coating. In some embodiments,the composition is formulated for delivery to the central nervoussystem, for example via intraspinal or intracranial delivery, orformulated to cross the blood brain barrier. In some embodiments, thecomposition is formulated to bypass the blood brain barrier. Suchformulations may be administered, for example, intranasally. Suchformulations may also be administered via the olfactory mute.

The present disclosure provides methods of identifying compositions thataffect the formation of microbially-induced amyloid. In some approaches,the methods comprise contacting a plurality of concentrations of amicrobial amyloid or a microbial amyloid precursor with a plurality ofconcentrations of α-synuclein and/or other mammalian amyloid ormammalian amyloid precursor in the presence of a composition, analyzingor measuring the formation of amyloid after the reaction set forthabove; and comparing said analysis or measurement to an analysis ormeasurement of a control, wherein said control comprises analyzing ormeasuring the formation of amyloid after the reaction set forth above inthe absence of said composition. In some methods and compositionsdisclosed herein, said microbial amyloid or microbial amyloid precursorcomprises CsgA. In some embodiments, the methods according to thepresent disclosure also comprise agitation during the contacting stepand/or prior to measurement.

In certain embodiments, said contacting of a plurality of concentrationsof a microbial amyloid or a microbial amyloid precursor (e.g., acomposition comprising CsgA) with a plurality of concentrations ofα-synuclein and/or other mammalian amyloid or mammalian amyloidprecursor is conducted in the presence of an indicator of amyloidformation. In some further embodiments, said indicator is a fluorescentindicator, a spin-labeled indicator, an enzyme, an antibody, or acolorimetric indicator. In some further embodiments, said indicator isThioflavin T (ThT). Where said indicator of amyloid formation is anantibody, the methods of the present disclosure provide that saidantibody may have specificity for aggregated α-synuclein and/or anothermammalian amyloid or mammalian amyloid precursor, and optionally may beconjugated to a fluorescent label, an enzyme, a colorimetric label, aspin label, a metal ion binding moiety, a nucleic acid, apolysaccharide, or a polypeptide. In some embodiments according to themethods of the present disclosure, CsgA and said α-synuclein and/orother such bacterial amyloid precursor and/or mammalianamyloid/mammalian amyloid precursor are each separately labeled.

In some embodiments according to the methods of the present disclosure,said contacting a plurality of concentrations of a microbial amyloid ora microbial amyloid precursor (e.g., a composition comprising CsgA) witha plurality of concentrations of α-Synuclein and/or other mammalianamyloid or mammalian amyloid precursor in the presence of a composition,analyzing or measuring the formation of amyloid after the reaction setforth above; and comparing said analysis or measurement to an analysisor measurement of a control, further comprises identifying or selectinga composition that alters or modulates or is suspected of altering ormodulating amyloid formation. In some embodiments, the methods describedherein further comprise identifying or selecting compositions thatreduce or enhance amyloid formation. In some embodiments, the methodsdescribed herein further comprise identifying or selecting compositionsthat reduce or enhance amyloid formation that also do not cross theblood brain barrier. The compounds identified by these methods, can beadministered to subjects identified or selected as a population thatwould benefit from receiving a compound that alters amyloid formation(e.g., a compound that reduces amyloid formation, preferably withoutcrossing the blood brain barrier). Such selected subjects may have beendiagnosed or evaluated for Parkinson's Disease. Lewy Body Dementia,incidental Lewy body disease. Lewy body variant of Alzheimer's disease,multiple system atrophy, or pure autonomic failure, or any combinationthereof.

The methods according to the present disclosure further contemplate amethod of making microbially-induced amyloid, comprising contacting aplurality of concentrations of a microbial amyloid or a microbialamyloid precursor with a plurality of concentrations of α-Synucleinand/or other mammalian amyloid or mammalian amyloid precursor in thepresence or absence of a composition; generating microbially-inducedamyloid; and analyzing or quantifying the microbially-induced amyloid.In some further embodiments, said microbial amyloid or microbial amyloidprecursor comprises CsgA. In some further embodiments, the methodsaccording to the present disclosure further comprise agitation duringsaid contacting or prior to measurement. In some further embodiments,said method is conducted in the presence of an indicator of amyloidformation. In some further embodiments, said indicator of amyloidformation may comprise a fluorescent indicator, a spin-labeledindicator, or a colorimetric indicator. In some embodiments, saidindicator said indicator is Thioflavin T (ThT). In some embodiments.CsgA and α-Synuclein, or other such bacterial amyloid/bacterial amyloidprecursor and mammalian amyloid/mammalian amyloid precursor are eachseparately labeled. In some embodiments, said amyloid formation isanalyzed or measured by internal fluorescence, by fluorescence of a dyeor label, by fluorescence resonance energy transfer, by fluorescencepolarization, by fluorescence polarization transfer, by UV/VisSpectroscopy, by magnetic resonance, by Raman scattering, by electronparamagnetic spin resonance, by light microscopy, by electronmicroscopy, by scanning tunneling microscopy, or by atomic forcemicroscopy.

In some embodiments according to the methods of the present disclosure,said composition to be present during said contacting of a plurality ofconcentrations of a microbial amyloid or a microbial amyloid precursor(e.g., a composition comprising CsgA) with a plurality of concentrationsof α-Synuclein and/or other mammalian amyloid or mammalian amyloidprecursor comprises a mixture of compounds. The composition may comprisetissue, bodily fluid or an extract thereof. In some embodiments, saidcomposition comprises feces, urine, blood, spinal fluid, or saliva, or acomponent thereof. In some embodiments, the composition comprises anextract from a natural product. In some further embodiments, the naturalproduct is an herb, a botanical substance, or foodstuff. In someembodiments, said natural product is a fungal tissue, legume, seed,berry, leaf, fruit, flower, plant root, plant stem, or plant bark. Insome embodiments, the composition may comprise one or more bacteria,bacterial extracts, lysates, conditioned culture media, lyophilizedbacteria, lyophilized lysates, lyophilized culture media, or anycombination thereof. In some embodiments, the composition may compriseone or more microbes, microbial extracts, lysates, conditioned culturemedia, lyophilized microbes, lyophized lysates, lyophilized culturemedia, or any combination thereof. In some embodiments, the methodsabove further comprise identifying or selecting compositions thatincrease or reduce amyloid formation, preferably compounds that also donot cross the blood brain barrier. The compounds identified by thesemethods, can be administered to subjects identified or selected as apopulation that would benefit from receiving a compound that altersamyloid formation (e.g., a compound that reduces amyloid formation,preferably without crossing the blood brain barrier). Such selectedsubjects may have been diagnosed or evaluated for Parkinson's Disease,Lewy Body Dementia, incidental Lewy body disease, Lewy body variant ofAlzheimer's disease, multiple system atrophy, or pure autonomic failure,or any combination thereof.

In some embodiments, the inhibitors of amyloid formation may be intendedfor administration systemically or locally to the enteric of centralnervous system. For example, inhibitors which are effective againstmammalian amyloid or mammalian amyloid precursor protein aggregation maybe useful in treatment of one or more of the amyloid disorders describedherein (e.g., one or more of the disorders of Table 3). Therefore, forsuch embodiments, the compositions comprising the inhibitors of amyloidformation may be formulated for parenteral administration, includingsystemic administration (e.g., intravenous, subcutaneous, intramuscular,intraperitoneal) or local administration (e.g., local injection near thevagus nerve, intraspinal injection, or intracranial injection). Fordelivery into the CNS, it is necessary for the inhibitors to passthrough the blood brain barrier. Therefore, in such embodiments, theinhibitors are preferably lipid soluble molecules, or may be modified toincrease lipid solubility, or may be co-administered with compounds thatenhance passage through the blood brain barrier (see, e.g.,WO2014076655A1, WO2012159052A2, WO1992018529A1).

The present disclosure also contemplates a kit comprising a microbialamyloid or a microbial amyloid precursor and α-Synuclein and/or othermammalian amyloid or mammalian amyloid precursor, being present in oneor more containers within said kit whereby the methods of the presentdisclosure may be practiced. In some embodiments, said microbial amyloidor microbial amyloid precursor comprises CsgA.

The present disclosure provides a method of inhibiting amyloid formationin a subject in need thereof, comprising administering to the subject acompound of the invention, as described herein, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof.

The present disclosure provides a method of preventing or treating adisorder associated with amyloid formation in a subject in need thereof,comprising administering to the subject a compound of the invention, asdescribed herein, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof.

The present disclosure provides a method of preventing or treating anamyloid disorder in a subject in need thereof, comprising administeringto the subject a compound of the invention, as described herein, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof.

In the methods of the present disclosure, the amyloid disorder or thedisorder associated with amyloid formation may be a neurologicaldisorder. The disorder may be Parkinson's disease (PD). Lewy bodydementia, multiple system atrophy, α-synucleinopathy, PD-associatedconstipation. PD-associated hyposmia, Huntington's Disease, Alexander'sDisease, amyotrophic lateral sclerosis (ALS), or Alzheimer's Disease.The disorder may be intestinal dysbiosis, intestinal hyperpermeability,irritable bowel syndrome (IBS), inflammatory bowel disease (IBD),ulcerative colitis or Crohns's disease.

The subject may suffer from gastrointestinal symptoms including one ormore of dysphagia, reduced gut motility, gastroparesis, constipation(including chronic constipation and chronic idiopathic constipation),small intestine bacterial overgrowth (SIBO), diarrhea, abdominal painand/or cramping, bloating, flatulence, hypersalivation (sialorrhea),anorectal dysfunction, dyssynergic defacation, and nausea. Thegastrointestinal symptoms may be associated with Parkinson's Disease orParkinsonism.

In certain embodiments, the amyloid disorder can be diagnosed bydetecting the presence or level of intestinal bacterial amyloidaggregates, the aggregates may comprise a bacterial CsgA protein. Incertain embodiments, the disorder can be diagnosed by detecting thepresence or level of intestinal bacterial genes and gene transcripts.

The methods of the present disclosure may further comprise detecting thepresence or level of a bacterial protein, such as CsgA, or amicroorganism that produces the bacterial protein, in an intestinalsample of the subject. In certain embodiments, the subject is selectedas in need of said prevention or treatment if the presence of thebacterial protein or the microorganism that produces the bacterialprotein is detected in the intestinal sample, or if the level of thebacterial protein or the microorganism that produces the bacterialprotein in the intestinal sample is greater than a predetermined levelor control.

The methods of the present disclosure may further comprise determining adecrease or absence of the intestinal amyloid aggregates following theadministration, or identifying the subject as displaying agastrointestinal symptom.

The methods of the present disclosure further provide a method oftreating or inhibiting an amyloid disorder (e.g., a neurologicaldisorder such as Parkinson's Disease, Lewy Body Dementia, incidentalLewy body disease. Lewy body variant of Alzheimer's disease, multiplesystem atrophy, or pure autonomic failure, or any combination thereof)in a tested subject comprising contacting a plurality of concentrationsof a microbial amyloid or a microbial amyloid precursor with a pluralityof concentrations of α-Synuclein and/or other mammalian amyloid ormammalian amyloid precursor, which may be obtained from a biologicalsample from said tested subject, in the presence or absence of acomposition; analyzing or measuring the formation of amyloid; andcomparing the analysis or measurement made with an analysis ormeasurement of a control, wherein said control may comprise analyzing ormeasuring the formation of amyloid in the absence of said composition orcomparison to a standard such as the amount or rate or formation ofamyloid from a healthy subject or a subject having amyloidosis (e.g., asubject suffering from Parkinson's Disease, Lewy Body Dementia,incidental Lewy body disease, Lewy body variant of Alzheimer's disease,multiple system atrophy, or pure autonomic failure, or any combinationthereof); and if the formation of amyloid in the presence of saidcomposition is increased relative to the formation of amyloid in theabsence of said composition or if the amount or rate or formation ofamyloid is the same or greater in the sample from the tested subject,for example, than the amount, rate, or formation of amyloid from thehealthy subject control or the control subject having amyloidosis,administering to said tested subject an effective amount of apharmaceutical composition suitable for inhibiting or treating saidamyloid disorder. In some further embodiments of these methods, saidmicrobial amyloid or microbial amyloid precursor comprises, consistsessentially of, or consists of CsgA.

In some embodiments, the methods as described herein further compriseidentifying or selecting said tested subject as one that would benefitfrom a treatment or inhibition of an amyloid disorder, and may furthercomprise identifying or selecting said subject as one at risk of orshowing symptoms of one or more of Parkinson's Disease, Lewy BodyDementia, incidental Lewy body disease. Lewy body variant of Alzheimer'sdisease, multiple system atrophy, pure autonomic failure, or anycombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-O are a series of graphs and images depicting that curli-drivenpathophysiology in mice requires functional amyloid formation. FIG. 1Ais a graph showing Crystal violet staining of biofilm growth bywild-type E. coli following 4 days in static culture, with indicatedconcentrations of epigallocatechin gallate (EGCG); data assessed byoptical density (OD). FIG. 1B is a graph showing in vitro αSynaggregation measured by Thioflavin T fluorescence during αSyn amyloidformation alone or in the presence of CsgA (25:1 molar ratio), with andwithout EGCG (50 μM) treatment. FIGS. 1C-L show results for germ-freeThy1-αSyn mice (ASO) mono-colonized with WT E. coli at 5-6 weeks of age,and given water alone (Vehicle: Veh) or treated with EGCG ad libitum indrinking water (+EGCG). FIG. 1C is a graph showing RNA was extractedfrom fecal pellets and csgA expression quantified by qRT-PCR, relativeto the E. coli reference gene rrsA. FIGS. 1D-H show assessment of motorfunction was assessed at 15-16 weeks of age by quantifying beamtraversal time (FIG. 1D), pole descent time (FIG. 1E), nasal adhesiveremoval time (FIG. 1F), hindlimb clasping score (FIG. 1G), and wirehangtests (FIG. 1H). FIG. 1I is a graph showing principal component analysisof compiled motor scores from tests in (FIGS. 1D-H). FIGS. 1J-K are aseries of graphs showing Proteinase K resistant αSyn aggregates(annotated with white arrows) in the substantia nigra imaged viaimmunofluorescence microscopy. Shown are vehicle-treated (FIG. 1J) andEGCG-treated mice (FIG. 1K). Fewer proteinase K resistant αSynaggregates were observed in the mouse treated with EGCG (FIG. 1K) thanin the vehicle-treated control (FIG. 1J). FIGS. 1L-M show quantificationof insoluble αSyn fibrils in the striatum (FIG. 1L) and ventral midbrain(FIG. 1M) by dot blot assay. FIGS. 1N-O are a series of graphs showingthin sections of brain were stained for Iba1 (microglia), 3D cellularreconstructions generated, and morphological characteristics quantifiedfrom microglia resident in the striatum (FIG. 1N) and substantia nigra(FIG. 1O), n=3 (FIGS. 1A, 1B, 1N, 1O), n=8 (FIG. 1C), n=10-11 (FIGS.1D-I), n=4 (FIGS. 1L-M). Points represent individuals, bars representthe mean and standard error. Data analyzed by one-way ANOVA with Tukeypost-hoc test for FIG. 1A, two-tailed Mann-Whitney for FIGS. C-K, ortwo-tailed t-test for FIG. 1L. For FIGS. 1A-1L *p≤0.05; **p≤0.01;***p≤0.001. Motor data are compiled from 2 independent cohorts.

FIGS. 2A-G are a series of graphs and images depicting thatmono-colonization with curli-sufficient bacteria induce increasedαSyn-dependent pathology and inflammatory responses in the brain.Germ-free (GF) wild-type (WT) or Thy1-αSyn (ASO) animals weremono-colonized with either wild-type, curli-sufficient E. coli (WT) orcurli-deficient E. coli (ΔcsgBAC). FIG. 2A is a graph showing total αSynin whole brain lysates quantified by ELISA. FIG. 2B is a graph showingquantification of insoluble αSyn fibrils in the striatum by dot blotassay. FIGS. 2C-D show quantification of TNFα (FIG. 2C) and IL-6 (FIG.2D) by ELISA from the striatum. FIGS. 2E-G show the results of stainingthin sections of brains derived from ASO mice. Sections were stained forIba1 (microglia), 3D cellular reconstructions generated, andmorphological characteristics quantified of microglia resident in thestriatum, n=3 (FIGS. 2A-B), n=6-7 (FIG. 2C, 2D), n=4 (FIGS. 2E-G)(averaged from 20-40 cells for diameters, or 5-7 cells for branching).Points represent individuals, bars represent the mean and standarderror. Data analyzed by one-way ANOVA with Tukey post-hoc test for FIGS.2A-D, or two-tailed t-test for FIGS. 2E and 2F. *p≤0.05; **p≤0.01;***p≤0.001; ****p≤0.0001.

FIGS. 2H-J are a series of graphs showing levels of csgA in human fecalsamples (FIG. 2H), in wild-type mice colonized with microbes derivedfrom persons with PD or matched controls (FIG. 2I), or in Thy1-αSyn(ASO) mice colonized with microbes derived from persons with PD ormatched controls (FIG. 2J). Consistent with these data, csgA ispredicted to be enriched in microbes derived from persons with PD.

FIGS. 3A-I are a series of graphs depicting that intestinal curlipromotes progressive synuclein-dependent pathophysiology.Conventionally-raised Thy1-αSyn (ASO) animals were injected intestinallywith 30 μg of synthetic CsgA hexamer (CsgA; N-QYGGNN-C) ornon-amyloidgenic peptide (N122A; N-QYGGNA-C). For FIGS. 3A-G, motor andGI function tested overtime at 0, 7, 21, and 70 days post-injection inthe beam traversal (FIG. 3A), pole descent (FIG. 3B), adhesive removal(FIG. 3C), hindlimb clasping score (FIG. 3D), wirehang (FIG. 3E), fecaloutput (at day 70) (FIG. 3F). FIG. 3G is a graph depicting principalcomponent analysis of compiled motor scores of FIGS. 3A-F. FIGS. 3H-Idepict quantification of insoluble αSyn fibrils in the striatum (FIG.3H) and ventral midbrain (FIG. 3I) by dot blot assay. n=8 (FIGS. 3A-G).n=4 (FIG. 3H). Points represent individuals, bars represent the mean andstandard error. Time courses analyzed by two-way ANOVA, with Sidakpost-hoc test for between group comparisons indicated above individualtime points, and brackets indicating significance between treatments.Data in (FIG. 3H) analyzed by two-tailed Mann-Whitney test. For FIGS.3A-I, *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.

FIGS. 4A-C are a series of graphs depicting fecal abundance ofamyloid-producing bacteria in humanized animals. Human samples fromprevious cohort (ENA Accession: PRJEB17694) were analyzed by PICRUSt toinfer abundance of csgA encoded within each population (FIG. 4A). Fecalpellets of Thy1-αSyn (ASO) mice receiving healthy-human derived fecalmicrobes enriched with either wild-type, curli-sufficient E. coli (WT)or curli-deficient E. coli (ΔcsgBAC) were analyzed by qPCR for rrsAabundance relative to 16s rRNA present in fecal bacterial DNA (FIG. 4B)and by qPCR analysis for csgA expression relative to rrsA in fecalbacterial RNA (FIG. 4C). n=5 (FIG. 4A), n=8 (FIGS. 4B-C). Pointsrepresent individuals, bars represent the mean and standard error. Datawere analyzed by two-tailed Mann-Whitney test. ***p≤0.001.

FIGS. 5A-J are a series of graphs depicting inhibition of functionalamyloid formation in accordance with some embodiments herein dampensprogressive motor deficits. Germ-free Thy1-αSyn mice (ASO) weremonocolonized with wild-type E. coli and treated with water alone(Vehicle, Veh) or given EGCG ad lib in drinking water (+EGCG). Motorfunction was assessed at 10, 12, and 15 weeks of age by quantifying beamtraversal time (FIG. 5A), pole descent time (FIG. 5B), nasal adhesiveremoval time (FIG. 5C), hindlimb clasping score (FIG. 5D), and wirehangtests (FIG. 5E). FIG. 5F depicts principal component analysis ofcompiled motor scores from FIGS. 5A-D. Thin sections of brain werestained for Iba1 (microglia) and morphological characteristicsquantified of microglia resident in the striatum (FIGS. 5G-H) andsubstantia nigra (FIGS. 51 -J). N=10-11 (FIGS. 5A-F), n=3 (FIGS. 5G-J)(averaged from 5-7 cells for branching). Bars represent the mean andstandard error. Time courses analyzed by two-way ANOVA, with Sidakpost-hoc test for between group comparisons indicated above individualtime points, and brackets indicating significance between treatments.Data in (FIGS. 5G-J) analyzed by two-tailed t-test. *p≤0.05; **p≤0.01;****p≤0.0001.

FIGS. 6A-6K are a series of graphs illustrating that the bacterialamyloid protein. CsgA, in accordance with some embodiments herein seedsαSyn fibrillization. In vitro biophysical analysis was conducted withpurified αSyn and CsgA proteins. FIG. 6A shows aggregation as measuredby Thioflavin T fluorescence over time during αSyn amyloid formationalone or in the presence of CsgA monomers (25:1 molar ratio, yellow).FIG. 6B shows time to reach exponential fibrillization, lag phase. FIGS.6C-H are a series of representative transmission electron micrographs ofαSyn alone (FIGS. 6C, 6F) or CsgA alone (FIGS. 6E, 6H), or incombination (FIGS. 6D, 6G), at 0 hours (FIGS. 6C-E) and 60 hours (FIGS.6F-H) post-aggregation. FIGS. 6I-K are a series of graphs illustratingcircular dichroism spectroscopic analysis of αSyn fibrillization aloneor in the presence of CsgA at 0, 12.5, and 60 hours post-aggregation.For FIG. 6A and FIG. 6B, n=3. Bars represent the mean and standarderror. Data are analyzed by two-tailed, t-test. **p≤0.01. Data arerepresentative of 2 independent trials.

FIGS. 7A-F are a series of graphs and transmission electron microscopeimages illustrating that CsgA seeds synuclein aggregation andpropagation through transient interactions. FIG. 7A is a graph showingThioflavin T fluorescence during αSyn amyloid formation alone or in thepresence of 5% seeds previously generated by addition of CsgA monomer toαSyn (as in FIG. 6A) or αSyn alone. FIGS. 7B-F are a series oftransmission electron micrograph of fibril structures generated by theaddition of above seeds and of seeds themselves. FIG. 7G is a graphshowing surface plasmon resonance measurements of surface immobilizedαSyn with additions of either CsgA monomer or seeds, or DOPS-DOPGcholesterol as positive control.

FIG. 8 is a graph illustrating an amyloid aggregation assay according tosome embodiments. Shown is the aggregation of α-Synuclein over time ismeasured by Thioflavin T (ThT) fluorescence, in the presence and absenceof CsgA.

FIG. 9 is a western blot for α-Synuclein in enteroendocrine (STC-1 cellline) cells treated with E. coli K12 or the ΔcsgBAC, curli-deficientstrain at an MOI of 10:1 for 4 hours.

FIG. 10 shows a method of synthesizing a compound of Formula (II).

FIG. 11 shows a method of synthesizing a compound of Formula (II).

FIG. 12 shows a method of synthesizing a compound of Formula (I).

FIG. 13 shows a method of synthesizing a compound of Formula (I).

DETAILED DESCRIPTION

The majority of cases of neurodegenerative diseases are idiopathic,which, conventionally, has made it difficult to identify the etiology ofmost such diseases. An emerging theory is that many neurodegenerativediseases start not in the brain or central nervous system (CNS), but inthe periphery and gradually migrate to the brain over the course of manyyears in a slow, progressive process. Still, the molecular etiology inthe periphery has been the subject of study. In the case of Parkinson'sDisease, it is known that constipation and hyposmia occur in manypatients often decades before the emergence of the stereotypical motorsymptoms that currently define Parkinson's Disease. Without beinglimited by theory, it is therefore contemplated that α-synucleinaggregation begins in the gastrointestinal (GI) tract and in theolfactory bulb, and that aggregated α-synuclein gradually progresses tothe brain in a prion-like propagative process. In this scenario, knownmore generally as Braak's Hypothesis, it is contemplated that analysisof the molecular mechanisms involved in these peripheral tissues canlead to non-intuitive, non-conventional approaches for preventing and/ortreating amyloid disorders, such as α-synucleinopathies, such asParkinson's Disease.

Without being limited by theory, one molecular mechanism contemplatedherein implicates bacterial amyloid as the seeding factor that nucleatesor otherwise leads to α-synuclein aggregation thereby initiating thepathological process that leads ultimately to Lewy body deposition andclinical manifestation of Parkinson's Disease and otherα-synucleinopathies. Bacterial amyloids are aggregated forms of secretedbacterial proteins and are thought to play a role in both bacterialadhesion to host cells and biofilm formation. In the right environmentand in the presence of host proteins prone to aggregation, it isbelieved, without being limited by theory, that bacterial amyloidsthemselves serve as a direct structural template for host proteinaggregation in a prion-like fashion. The bacterial chaperone machineryresponsible for driving bacterial amyloid aggregation may also use thehost protein as a substrate and thereby facilitate host proteinaggregation into amyloid structures. Once aggregated, the host proteinaggregation is perpetuated in a prion-like fashion through the entericnervous system over the course of many years. Ultimately, theseaggregates spread into brain tissue and result in the stereotypicalclinical symptoms of Parkinson's Disease. This effect may also result inthe development of other amyloid-driven diseases such as Alzheimer'sdisease, in which aggregation of the host proteins A-beta and/or tau areimplicated. Consistent with this, analysis of current publicly-availablehuman microbiome datasets reveals increased representation of thecurli-associated csgA gene from E. coli in persons with Parkinson'sDisease, and transplantation of fecal microbes from PD patients intogerm-free (GF) wild-type or ASO mice results in greater csgA abundancecompared to microbiomes from healthy controls, based on PICRUSt imputedanalysis of 16s rRNA sequences (See Example 7). It is shown herein thatintestinal amyloid aggregates can lead to symptoms associated withParkinson's Disease and other amyloid disorders (See Examples 6-8), andthat treating these animals with compounds that inhibit and/or disruptamyloid aggregates can ameliorate these symptoms associated withParkinson's Disease and other amyloid disorders (See Example 10). Insome embodiments, a method for inhibiting, ameliorating, reducing thelikelihood, delaying the onset of, treating, and/or preventing theamyloid disorder comprising administering a composition (e.g., apharmaceutical composition) comprising a compound of the invention, forexample any of the compounds of Tables 1 and 2 or Table 4 (infra). Insome embodiments, the composition comprises or consists essentially ofone or more compounds selected from the group consisting of thecompounds identified by a compound activity range of “+++” in any columnof Table 4. In some embodiments, the composition comprises or consistsessentially of one or more compounds selected from the group consistingof the set of compounds identified by a compound activity range of “++”or “+++” in any column of Table 4. In some embodiments, the compositioncomprises or consists essentially of one or more compounds selected fromthe group consisting of the set of compounds identified by a compoundactivity range of “+”. “++,” or “+++” in any column of Table 4. In someembodiments, the composition comprises or consists essentially of one ormore compounds selected from the group consisting of the compoundsidentified by a compound activity range of “+++” in the “αSyn” column ofTable 4. In some embodiments, the composition comprises or consistsessentially of one or more compounds selected from the group consistingof the set of compounds identified by a compound activity range of “++”or “+++” in the “αSyn” column of Table 4. In some embodiments, thecomposition comprises or consists essentially of one or more compoundsselected from the group consisting of the set of compounds identified bya compound activity range of “+”, “++,” or “+++” in the “αSyn” column ofTable 4. In some embodiments, the composition comprises or consistsessentially of one or more compounds selected from the group consistingof the compounds identified by a compound activity range of “+++” in the“CsgA-seeded αSyn” column of Table 4. In some embodiments, thecomposition comprises or consists essentially of one or more compoundsselected from the group consisting of the set of compounds identified bya compound activity range of “++” or “+++” in the “CsgA-seeded αSyn”column of Table 4. In some embodiments, the composition comprises orconsists essentially of one or more compounds selected from the groupconsisting of the set of compounds identified by a compound activityrange of “+”. “++.” or “+++” in the “CsgA-seeded αSyn” column of Table4. In some embodiments, the composition comprises or consistsessentially of one or more compounds selected from the group consistingof the compounds identified by a compound activity range of “+++” in the“CsgA” column of Table 4. In some embodiments, the composition comprisesor consists essentially of one or more compounds selected from the groupconsisting of the set of compounds identified by a compound activityrange of “++” or “+++” in the “CsgA” column of Table 4. In someembodiments, the composition comprises or consists essentially of one ormore compounds selected from the group consisting of the set ofcompounds identified by a compound activity range of “+”, “++” or “+++”in the “CsgA” column of Table 4. In some embodiments, the subject isselected as in need of the composition by detecting a presence and/orlevel of aggregates in an intestinal sample of the subject, such as afecal sample. A presence or level of intestinal aggregates greater thana negative control (for example, fecal sample of a healthy controlsubject, or control subject known not to have an amyloid disorder) canindicate that the subject is in need of the composition.

In some embodiments, detecting the presence and/or level of intestinalaggregates in a sample of the subject comprises detecting a presenceand/or level of a bacterial protein in the sample, for example acurli-associated protein, such as CsgA. In some embodiments, detectingthe presence and/or level of intestinal aggregates in a sample of thesubject comprises detecting a level of a bacteria that produces anamyloid in the sample, for example a curli-associated protein, such asCsgA. For example, a bacterial amyloid can be detected directly, or anucleic acid encoding the amyloid can be detected in the sample, thusindicating a presence of amyloid-producing bacteria in the subject'sgastrointestinal tract. Examples of amyloid-producing bacteria caninclude CsgA-producing Enterobacteriaceae such as E. coli.

Amyloids are produced in the gastrointestinal tract by members of thegastrointestinal microbiota, such as 1E. coli and some otherProteobacteria. These microbial amyloids may interact with cells withwhich they are in contact in the gastrointestinal tract and affectα-synuclein expression and/or α-synuclein aggregation. The STC-1 cellline was derived from tumors of the mouse small intestine and possessesmany features of native gastrointestinal enteroendocrine cells (McCarthyet al. (2015), STC-1 Cells. In: Verhoeckx K, et al. (eds) The Impact ofFood Bioactives on Health. Springer, Cham.). In an in vitro assaywherein α-synuclein expression by STC-1 cells was determined by Westernblot, exposure to an E. coli strain expressing wild-type CsgA resultedin a notable increase in α-synuclein expression, while exposure to anisogenic mutant in which csgA was deleted had little effect onα-synuclein levels (See FIG. 9 and Example 33). Thus, while the exactmechanisms by which CsgA affected α-synuclein expression are unclear,CsgA can interact with enteroendocrine-like cells of thegastrointestinal tract and cause α-synuclein over-expression in vitro,suggesting that similar effects may take place in vivo when pathogenicmicrobial amyloids contact enteroendocrine cells or other cells in thegastrointestinal tract. While mouse α-synuclein is generally notobserved to aggregate, over-expression of human α-synuclein may lead toaggregation that in turn impairs cell function, propagates in aprion-like fashion to adjacent cells in the gastrointestinal tract andenteric nervous system, and has detrimental effects on gastrointestinalfunction. These negative effects can include one or more of intestinaldysbiosis, intestinal hyperpermeability, irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), ulcerative colitis and/or Crohn'sdisease. These disorders can be associated with one or more symptoms,including dysphagia, reduced gut motility, gastroparesis, constipation(including chronic constipation and chronic idiopathic constipation),small intestinal bacterial overgrowth (SIBO), diarrhea (includingchronic diarrhea), abdominal pain and/or cramping, bloating, flatulence,and nausea. In some cases, neurological and gastrointestinal symptoms ofamyloid disorders can be linked. For example, in Parkinson's Disease andParkinsonism (a clinical syndrome characterized by tremor, bradykinesia,rigidity, and postural instability) decreased levels of dopamine canlead to the neurological symptom of dyskinesia and the gastrointestinalsymptom of chronic idiopathic constipation. Thus, treatments whichimprove gut motility, including the methods of the invention, canimprove dopamine absorption in the gut and, thereby, reduce dyskinesia.Therefore, treatments that manage constipation (or, more generally,intestinal dysbiosis or intestinal hyperpermeability) can slow theprogression of motor symptoms of Parkinson's Disease as well asincreasing “on-time” periods of adequate control of Parkinson's Diseasesymptoms.

Consistent with the ability of STC-1 cells to respond to E. coli CsgA invitro, gastrointestinal cells have been observed to sense and respond tomicrobial amyloids. For example, Salmonella enterica CsgA has been shownto modulate gastrointestinal permeability in mice via activation of theTLR2/PI3K pathway. Additionally, Tukel discloses in U.S. Pat. No.9,814,756 a method for modulating gastrointestinal permeability viaadministration of variants of CsgA and/or CsgB. Tukel discloses in U.S.Pat. No. 9,814,756 decreasing permeability of epithelium of the smallintestine or large intestine by administering a composition comprising,inter alia, an isolated curli fibril having epitheliumpermeability-reducing activity such as (i) a CsgA polypeptide variantwhich differs from a naturally occurring CsgA polypeptide in that from 1to 5 amino acids have been substituted, deleted or added; (ii) a CsgBpolypeptide variant which differs from a naturally occurring CsgBpolypeptide in that from 1 to 5 amino acids have been substituted,deleted or added; or (iii) a combination of said CsgA polypeptidevariant and said CsgB polypeptide variant. Thus, while there may beadditional mechanisms by which microbial amyloids interact withgastrointestinal cells, at least one such mechanism is contemplatedherein.

The present disclosure relates to methods and compositions for thetreatment, amelioration, or prevention of amyloid disorders. Disclosedherein are compositions and methods, which alter the ability ofbacterial amyloid to promote aggregation and amyloid formation of theeukaryotic protein α-synuclein. Said alterations may include alterationsin the extent, rate of formation, stability, and/or rate ofdisaggregation of microbially induced amyloid, or any combinationthereof. Further disclosed herein are compositions (e.g., comprisingcompounds of the invention) and methods useful for the treatment orinhibition of neurodegenerative diseases, as well as, compositions andmethods useful for the prevention or amelioration of the progression ofneurodegenerative diseases. Further disclosed herein are compositionsand methods useful for the treatment or inhibition of gastrointestinaldysfunction related to neurodegenerative diseases. Additionallydisclosed herein are methods for studying the molecular etiology ofmammalian amyloid diseases and the molecular link between bacterialamyloid production and mammalian amyloid production. According to themethods of the present disclosure, said neurodegenerative diseasesand/or mammalian amyloid diseases may comprise one or more ofParkinson's disease (PD). Lewy body dementia, multiple system atrophy,and all other α-synucleinopathies, PD-associated constipation.PD-associated hyposmia, Huntington's Disease. Alexander's Disease,amyotrophic lateral sclerosis (ALS), Alzheimer's Disease and otherdiseases in which amyloids are implicated. In some embodiments, thecomposition comprises, consists essentially of, or consists of acompound of the invention as described herein. The present disclosurefurther relates to methods that facilitate the evaluation of aggregationand dis-aggregation of both host and bacterial amyloid proteins. Methodsof the present disclosure are also useful for identifying drugcandidates that affect these processes.

It is contemplated that in some embodiments, a composition comprising,consisting essentially of, or consisting of a compound of the inventionas described herein is useful in preventing α-synuclein aggregation, theseeding of α-synuclein aggregation by CsgA or other microbial amyloids,and the formation of microbial amyloids that may seed α-synucleinaggregation in vivo, and these compounds may thus be useful inpreventing or treating Parkinson's Disease and/or otherα-synucleinopathies (See, e.g., Example 32 and Table 3).

It is contemplated that in some embodiments, a composition comprising,consisting essentially of, or consisting of a compound of the inventionas described herein may be useful in preventing α-synuclein aggregationwith or without seeding by microbial amyloids and thereby may havebenefit in preventing or treating α-synucleinopathies independent ofmicrobial amyloids (See, e.g., Example 32 and Table 3).

It is contemplated that in some embodiments, a composition comprising,consisting essentially of, or consisting of a compound of the inventionas described herein may be useful in preventing α-synuclein aggregationseeded by microbial amyloids and thereby have therapeutic benefit, forexample if dosed at sites where microbial amyloids may be abundant, suchas the gastrointestinal tract (See, e.g., Example 32 and Table 3).

It is contemplated that in some embodiments, a composition comprising,consisting essentially of, or consisting of a compound of the inventionas described herein may have therapeutic benefit in Parkinson's Diseaseand other α-synucleinopathies. Without being limited by theory, thisbenefit may be due to these compounds' inhibition of aggregation ofα-synuclein and/or microbial amyloids. It is further contemplated thatfor compounds in which more than one type of aggregation is inhibited,these inhibitory effects may be additive or synergistic. (See, e.g.,Example 32 and Table 3).

Accordingly, provided herein are compounds (i.e., compounds of theinvention) that are useful in inhibiting, ameliorating, reducing thelikelihood, delaying the onset of, treating, and/or preventing anamyloid disorders for example, any of the amyloid disorders of Table 4(infra), such as an α-synucleinopathy. Parkinson's Disease, Lewy BodyDementia, incidental Lewy body disease, Lewy body variant of Alzheimer'sdisease, multiple system atrophy, pure autonomic failure, or acombination of two or more of the listed items.

Compounds of the Invention

In one aspect, provided herein is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof.

Formula (I), embodiments of Formula (I) provided herein, andpharmaceutically acceptable salts thereof, are defined as follows:

L¹ is a divalent moiety selected from a bond and —N(R⁵)(C═O)—.

L² is a divalent moiety selected from —(C═O)— and —(C═O)O—.

G¹ is a monovalent moiety selected from —N(R⁵)₂ and R⁶. In certainembodiments, G¹ is —NH₂. In certain embodiments, G¹ is —NHR⁵. In certainembodiments, G¹ is —N(R⁵)₂. In certain embodiments, G¹ is R⁶.

G² is a monovalent moiety selected from —H, —N(R⁵)₂, and —N(R⁵)(R⁶). Incertain embodiments, G² is —H. In certain embodiments, G² is —NH₂. Incertain embodiments, G² is —NHR⁵. In certain embodiments, G² is —N(R⁵)₂.In certain embodiments, G² is —NHR⁶. In certain embodiments, G² is—N(R⁵)(R⁶).

Each R⁵ is a monovalent moiety independently selected from —H and alkyl.In certain embodiments, R⁵ is substituted C₁₋₆ alkyl. In certainembodiments, R⁵ is unsubstituted C₁₋₆ alkyl. In certain embodiments, R⁵is methyl. In certain embodiments, R³ is —H.

Each R⁶ is aryl substituted with n instances of R⁷. For each R⁶, n is 0,1, 2, 3, or 4. In certain embodiments, n is 0. In certain embodiments, nis 1. In certain embodiments, n is 2. In certain embodiments, n is 3. Incertain embodiments, n is 4.

Each R⁷ is independently selected from —F, —OH, and —O(alkyl). Incertain embodiments, each R⁷ is independently selected from —F, —OH, and—OCH₃. In certain embodiments, each R⁷ is independently selected from—OH and —OCH₃. In certain embodiments, each R⁷ is —OH. In certainembodiments, each R⁷ is —OCH₃.

In certain embodiments, Formula (I) is of Formula (I-a):

or a pharmaceutically acceptable salt thereof.

In certain embodiments of Formula (I-a):

G¹ is R⁶;

G² is —NH₂ or —N(R⁵)(R⁶);

R⁵ is —H or —CH₃;

R⁶ is phenyl substituted with n instances of R⁷;

R⁷ is independently, for each occurrence. —F, —OH or —OCH₃; and

n is 0, 1, 2, 3, or 4.

In certain embodiments, Formula (I) is of Formula (I-b):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, Formula (I-b) is of Formula (I-b-1) or Formula(I-b-2):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of Formula (I), (I-a), (I-b),(I-b-1), or (I-b-2), or a pharmaceutically acceptable salt thereof, has0-6 instances of R⁷. In certain embodiments, the compound has 1-6instances of R⁷. In certain embodiments, the compound has 2-6 instancesof R⁷. In certain embodiments, the compound has 3-6 instances of R⁷. Incertain embodiments, the compound has 1-3 instances of R⁷. In certainembodiments, the compound has 2-4 instances of R⁷. In certainembodiments, the compound has 2 instances of R⁷. In certain embodiments,the compound has 3 instances of R⁷. In certain embodiments, the compoundhas 4 instances of R⁷. In certain embodiments, the compound has 5instances of R⁷. In certain embodiments, the compound has 6 instances ofR⁷.

In certain embodiments of the compound of Formula (I), (I-a), (I-b),(I-b-1), or (I-b-2), or a pharmaceutically acceptable salt thereof, eachR⁶ is independently selected from:

In certain embodiments of the compound of Formula (I), (I-a), (I-b),(I-b-1), or (I-b-2), or a pharmaceutically acceptable salt thereof, eachR⁶ is independently selected from:

In certain embodiments, it is provided that the compound of Formula (I),(I-a), (I-b), (I-b-1), or (I-b-2):

(a) comprises at least one instance of R⁶; and/or

(b) comprises at least three instances of R⁷; and/or

(c) is not one of the following compounds:

In certain embodiments, the compound of Formula (I), (I-a), (I-b),(I-b-1), or (I-b-2) is selected from the compounds of Table 1, andpharmaceutically acceptable salts thereof.

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

In another aspect, provided herein is a compound of Formula (II):

or a pharmaceutically acceptable salt thereof.

Formula (II), embodiments of Formula (II) provided herein, andpharmaceutically acceptable salts thereof, are defined as follows.

L¹ is a bond.

L² is a divalent moiety selected from a bond, —CH₂—, —(C═O)—, —(C═O)O—,and —NR²(C═O)—.

G¹ is a monovalent moiety selected from —H and R³. In certainembodiments, G¹ is —H. In certain embodiments, G¹ is R³.

G² is a monovalent moiety selected from —H, —N(R²)₂, —N(R²)(R³), and R³.In certain embodiments, G² is —H. In certain embodiments, G² is —N(R²)₂.In certain embodiments, G² is —NH₂. In certain embodiments, G² is—N(C₁₋₆ alkyl)₂. In certain embodiments, G² is —N(R²)(R³). In certainembodiments, G² is R³.

R is a monovalent moiety selected from —H and alkyl. In certainembodiments, R is substituted C₁₋₆ alkyl. In certain embodiments, R isunsubstituted C₁₋₆ alkyl. In certain embodiments, R is methyl. Incertain embodiments, R is —H.

Each R¹ is independently selected from —F, —OH, and —O(alkyl). Incertain embodiments, each R¹ is independently selected from —F, —OH, and—OCH₃. In certain embodiments, each R¹ is independently selected from—OH and —OCH₃. In certain embodiments, each R¹ is —OH. In certainembodiments, each R¹ is —OCH₃.

Each R² is a monovalent moiety independently selected from —H and alkyl.In certain embodiments, R² is substituted C₁₋₆ alkyl. In certainembodiments, R² is unsubstituted C₁₋₆ alkyl. In certain embodiments, R²is methyl. In certain embodiments, R² is —H.

Each R³ is aryl substituted with n instances of R⁴. For each R³, n is 0,1, 2, 3, or 4. In certain embodiments, n is 0. In certain embodiments, nis 1. In certain embodiments, n is 2. In certain embodiments, n is 3. Incertain embodiments, n is 4.

Each R⁴ is independently selected from —F, —OH, and —O(alkyl). Incertain embodiments, each R⁴ is independently selected from —F, —OH, and—OCH₃. In certain embodiments, each R¹ is independently selected from—OH and —OCH₃. In certain embodiments, each R⁴ is —OH. In certainembodiments, each R⁷ is —OCH₃.

m is 0, 1, 2, 3, or 4. In certain embodiments, m is 0. In certainembodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 3. In certain embodiments, m is 4.

In certain embodiments,

L¹ is a bond;

L² is a bond, —CH₂—, —(C═O)—, —(C═O)O—, or —NR²(C═O)—;

G¹ is —H or R³;

G² is —N(R²)(R³) or R³;

R is H or —CH₃;

R¹ is —OH or —OCH₃;

R² is —H or —CH₃;

R³ is phenyl substituted with m instances of R⁴;

R⁴ is independently, for each occurrence, —F, —OH or —OCH₃;

n is, for each occurrence 0, 1, 2, 3, or 4; and

m is 0, 1, 2, 3, or 4.

In certain embodiments, Formula (II) is of Formula (II-a) or Formula(II-b):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, Formula (II-a) is of Formula (II-a-1) or Formula(II-a-2):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, Formula (II-b) is of Formula (II-b-1) or Formula(II-b-2):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of Formula (II), (II-a), (II-a-1)(II-a-2), (II-b), (II-b-1), (II-b-2), or a pharmaceutically acceptablesalt thereof, has 0-6 instances of R⁴. In certain embodiments, thecompound has 1-6 instances of R⁴. In certain embodiments, the compoundhas 2-6 instances of R⁴. In certain embodiments, the compound has 3-6instances of R⁴. In certain embodiments, the compound has 1-3 instancesof R⁴. In certain embodiments, the compound has 2-4 instances of R⁴. Incertain embodiments, the compound has 2 instances of R⁴. In certainembodiments, the compound has 3 instances of R⁴. In certain embodiments,the compound has 4 instances of R⁴. In certain embodiments, the compoundhas 5 instances of R⁴. In certain embodiments, the compound has 6instances of R⁴.

In certain embodiments of the compound of Formula (I), (II-a), (II-a-1),(II-a-2), (II-b), (II-b-1), (II-b-2), or a pharmaceutically acceptablesalt thereof, the sum of all instances of m and n is 0-9. In certainembodiments, the sum is 1-9. In certain embodiments, the sum is 2-9. Incertain embodiments, the sum is 3-9. In certain embodiments, the sum is4-9. In certain embodiments, the sum is 5-9. In certain embodiments, thesum is 6-9. In certain embodiments, the sum is 1-6. In certainembodiments, the sum is 2-6. In certain embodiments, the sum is 3-6. Incertain embodiments, the sum is 1-3. In certain embodiments, the sum is2. In certain embodiments, the sum is 3. In certain embodiments, the sumis 4. In certain embodiments, the sum is 5. In certain embodiments, thesum is 6. In certain embodiments, the sum is 7. In certain embodiments,the sum is 8. In certain embodiments, the sum is 9.

In certain embodiments of the compound of Formula (II), (II-a),(II-a-1), (II-a-2), (II-b), (II-b-1), (II-b-2), or a pharmaceuticallyacceptable salt thereof, each R³ is independently selected from:

In certain embodiments of the compound of Formula (II), (II-a),(II-a-1), (II-a-2), (II-b), (II-b-1), (II-b-2), or a pharmaceuticallyacceptable salt thereof, each R³ is independently selected from:

In certain embodiments of the compound of Formula (II), (II-a),(II-a-1), (II-a-2), (II-b), (II-b-1), or (II-b-2), it is provided that:

(a) the compound comprises at least one instance of R³; and/or(b) the sum of all instances of m and n is at least three; and/or(c) the compound does not have the structure:

In certain embodiments, the compound of Formula (II), (II-a), (II-a-1),(II-a-2), (II-b), (II-b-1), or (II-b-2) is selected from the compoundsof Table 2, and pharmaceutically acceptable salts thereof.

TABLE 2

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

The compounds described above, and pharmaceutically acceptable saltsthereof, may be referred to collectively as compounds of the invention.

The term “alkyl” refers to a radical of a straight-chain or branchedsaturated hydrocarbon group having from 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), propyl(C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl,sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl,neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C₆) (e.g.,n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇),n-octyl (C₈), and the like. Unless otherwise specified, each instance ofan alkyl group is independently unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents(e.g., halogen, such as F). In certain embodiments, the alkyl group isan unsubstituted C₁₋₁₀ alkyl (such as unsubstituted C₁₋₆ alkyl, e.g.,—CH₃ (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g.,unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)),unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu),unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl(sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certainembodiments, the alkyl group is a substituted C₁₋₁₀ alkyl (such assubstituted C₁₋₆ alkyl, e.g., —CH₂F, —CHF₂, —CF₃ or benzyl (Bn)). Analkyl group may be branched or unbranched.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14 π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents (e.g., —F, —OH or—O(C₁₋₆ alkyl). In certain embodiments, the aryl group is anunsubstituted C₆₋₁₄ aryl. In certain embodiments, the aryl group is asubstituted C₆₋₁₄ aryl.

“Solvate” has its customary and ordinary meaning as understood by one ofskill in the art in view of this disclosure. It refers to the compoundformed by the interaction of a solvent and an active pharmaceuticalingredient (or API), a metabolite, or salt thereof. Suitable solvatesare pharmaceutically acceptable solvates including hydrates.

The terms “decrease”, “reduced”, “reduction”, “inhibit” or “disrupt” areall used herein to mean a decrease by a statistically significantamount. In some embodiments, “reduce,” “reduction”, “decrease”,“inhibit” or “disrupt” typically means a decrease by at least 10% ascompared to a reference level (e.g. the absence of a given treatment)and can include, for example, a decrease by at least about 10%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or more. As used herein, “reduction” or “inhibition” does not encompassa complete inhibition or reduction as compared to a reference level.“Complete inhibition” is a 100% inhibition as compared to a referencelevel. A decrease can be preferably down to a level accepted as withinthe range of normal for an individual without a given disorder.

An “effective amount” or “effective dose” of a compound (e.g., acompound of the invention) or composition containing such compound,refers to the amount sufficient to achieve a desired biological and/orpharmacological effect, e.g., when delivered to a cell or organismaccording to a selected administration form, mute, and/or schedule. Thephrases “effective amount” and “therapeutically effective amount” areused interchangeably. As will be appreciated by those of ordinary skillin this art, the absolute amount of a particular compound or compositionthat is effective may vary depending on such factors as the desiredbiological or pharmacological endpoint, the agent to be delivered, thetarget tissue, etc. Those of ordinary skill in the art will furtherunderstand that an “effective amount” may be administered to a subjectin a single dose, or through use of multiple doses, in variousembodiments.

Pharmaceutical Compositions, Formulation, Administration and Dosing

In another aspect, provided herein is a pharmaceutical compositioncomprising a compound of the invention and a pharmaceutically acceptablecarrier. Pharmaceutical compositions described herein are useful forinhibiting amyloid formation.

In certain embodiments, the pharmaceutical composition is formulated fordelivery outside of the systemic circulation of a subject. In certainembodiments, the pharmaceutical composition is formulated for deliveryto the central nervous system of a subject. Said composition may beformulated for enteric or intranasal delivery, for example, and/or saidcompositions may further be formulated for controlled release within thelower intestine or colon. The aforementioned compositions may comprisean enteric-coated capsule, tablet, soft-gel, spray dried powder, polymermatrix, hydrogel, enteric-coated solid, crystalline solid, amorphoussolid, glassy solid, coated micronized particle, liquid, nebulizedliquid, aerosol, or microcapsule.

In certain embodiments, the pharmaceutical composition is formulated fororal administration. In certain embodiments, the pharmaceuticalcomposition is formulated for administration by injection. The injectionmay be intravenous, subcutaneous, intramuscular, intraperitoneal,intraspinal or intracranial.

The pharmaceutical composition an effective amount of one or morecompounds of the invention. The effective amount is sufficient toachieve one or more desired biological and/or pharmacological effects,e.g., the disruption or inhibition of the formation of amyloidaggregates, the treatment or prevention of a neurological disorder, orsymptoms of a neurological disorder, or the treatment or prevention of agastrointestinal disorders, or symptoms of a gastrointestinal disorder.

“Administering” has its customary and ordinary meaning as understood byone of skill in the art in view of this disclosure. It refers toproviding a pharmaceutical agent, dietary supplement, or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administration. Administration of thecompounds disclosed herein or the pharmaceutically acceptable saltsthereof can be via any of the accepted modes of administration foragents that serve similar utilities including, but not limited to,orally, intraperitoneally, or rectally. Oral administrations arecustomary in administering the compositions that are the subject of thepreferred embodiments. However, in some embodiments, the compositions tobe administered according to the methods of the present disclosure areadministered rectally, such as by enema or suppository. In someembodiments, administration of the compounds may occur outside the body,for example, by apheresis or dialysis.

The term “agent” has its customary and ordinary meaning as understood byone of skill in the art in view of this disclosure. It includes anysubstance, molecule, element, compound, entity, or a combinationthereof. It includes, but is not limited to, e.g., protein, polypeptide,peptide or mimetic, small organic molecule, polysaccharide,polynucleotide, polymer, resin, organic or inorganic microparticle,organic or inorganic nanoparticle, and the like. It can be a naturalproduct, a synthetic compound, or a chemical compound, or a combinationof two or more substances.

In some embodiments, the methods of the present disclosure contemplatethe administration of one or more compositions useful for theamelioration or treatment of one or more neurological disordersassociated with amyloid formation. Said compositions can be formulatedinto pharmaceutical compositions and/or dietary supplements for use intreating, inhibiting, or ameliorating a neurological disease orneurological disorder associated with amyloid formation such asParkinson's disease (PD), Lewy body dementia, multiple system atrophy,and all other α-synucleinopathies. PD-associated constipation,PD-associated hyposmia. Huntington's Disease. Alexander's Disease,amyotrophic lateral sclerosis (ALS), and/or Alzheimer's Disease and/orother diseases in which amyloids are implicated. Standard pharmaceuticaland/or dietary supplement formulation techniques are used, such as thosedisclosed in Remington's The Science and Practice of Pharmacy, 21st Ed.,Lippincott Williams & Wilkins (2005), incorporated herein by referencein its entirety. Accordingly, some embodiments include pharmaceuticaland/or dietary supplement compositions comprising: (a) a safe andtherapeutically effective amount of one or more compounds describedherein, or pharmaceutically acceptable salts thereof; and (b) apharmaceutically acceptable carrier, diluent, excipient or combinationthereof.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” has its customary and ordinary meaning asunderstood by one of skill in the art in view of this disclosure. Itincludes any and all solvents, diluents, emulsifiers, binders, buffers,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like, or any other suchcompound as is known by those of skill in the art to be useful inpreparing pharmaceutical formulations. The use of such media and agentsfor pharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. In addition, various adjuvants such as arecommonly used in the art may be included. These and other such compoundsare described in the literature, e.g., in the Merck Index, Merck &Company. Rahway, N.J. Considerations for the inclusion of variouscomponents in pharmaceutical compositions are described, e.g., in Gilmanet al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th Ed., Pergamon Press.

Some examples of substances, which can serve aspharmaceutically-acceptable carriers or components thereof in accordancewith methods and compositions of some embodiments herein, are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe TWEENS; wetting agents, such as sodium lauryl sulfate; coloringagents; flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and/or phosphatebuffer solutions, or any combination thereof.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the one or more compounds for administration asdescribed herein can be determined by the way the compound is to beadministered.

In addition, the present disclosure includes compositions comprisingvarious salts, esters, hydrates, prodrugs, fluorinated analogs, orisotopically substituted analogs, including deuterated forms, of thecompounds described herein.

As used herein, “systemic circulation” has its customary and ordinarymeaning as understood by one of skill in the art in view of thisdisclosure. It refers to circulation within the blood or circulatorysystem of a subject.

As used herein, “enteric coating” has its customary and ordinary meaningas understood by one of skill in the art in view of this disclosure. Itrefers to a pharmaceutical excipient coating or placed around a particlewhich, by control of its solubility or timing of dissolution, increasesthe likelihood that said particle will be protected from solvent untilits arrival in a desired portion of the gastrointestinal tract, forexample, by conferring resistance to stomach acid or by having highersolubility at neutral or basic pH. Representative enteric coatingsinclude, for example, those described in Remington's The Science andPractice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005).Exemplary enteric coatings include but are not limited to, shellac,sodium alginate, zein, cellulose acetate trimellitate, methylmethacrylate-methacrylic acid copolymer, polyvinyl acetate phthalate,polylactic acid, polylactic-co-glycolic acid, hypromellose acetate,hypromellose acetate succinate. Hydroxypropyl methyl cellulosephthalate, Cellulose acetate succinate. Cellulose acetate phthalate.Methyl acrylate-methacrylic acid copolymer, polyvinyl acetate phthalate.Opadry®, and others as are known in the art of drug delivery andformulation. In accordance with method and compositions of someembodiments, the composition comprising a compound as described hereinfurther comprises an enteric coating.

The term “gut selective” as used herein has its customary and ordinarymeaning as understood by one of skill in the art in view of thisdisclosure. It refers to a composition or formulation that is releasedin the gut of a subject, and preferably is not absorbed, or ifabsorption occurs, does not enter the systemic circulation.

The term “intrinsically enteric” as used herein has its customary andordinary meaning as understood by one of skill in the art in view ofthis disclosure. With reference to a pharmaceutical formulation refersto a composition which innately has the ability to preventdisintegration or release in the gastric environment.

A composition for administration to a subject as described herein ispreferably provided in a unit dosage form. As used herein, a “unitdosage form” has its customary and ordinary meaning as understood by oneof skill in the art in view of this disclosure. It refers to acomposition containing an amount of a compound that is suitable foradministration to a subject, in a single dose, according to good medicalpractice. The preparation of a single or unit dosage form however, doesnot imply that the dosage form is administered once per day or once percourse of therapy. A unit dosage form may comprise a single daily doseor a fractional sub-dose wherein several unit dosage forms are to beadministered over the course of a day in order to complete a daily dose.According to the present disclosure, a unit dosage form may be givenmore or less often that once daily, and may be administered more thanonce during a course of therapy. Such dosage forms may be administeredin any manner consistent with their formulation, including orally,rectally, nasally, and/or parenterally. While single administrations arespecifically contemplated, the compositions administered according tothe methods described herein may also be administered as a continuousinfusion or via an implantable infusion pump.

The methods as described herein may utilize any of a variety of suitableforms for a variety of routes for administration, for example, for oral,nasal, rectal, or parenteral routes of administration. Depending uponthe particular route of administration desired, a variety ofpharmaceutically-acceptable carriers well-known in the art may be used.Pharmaceutically-acceptable carriers include, for example, solid orliquid fillers, diluents, hydrotropes, surface-active agents, andencapsulating substances. Optional pharmaceutically-active materials maybe included, which do not substantially interfere with the activity ofthe one or more compounds in the formulation. The amount of carrieremployed in conjunction with the compound is sufficient to provide apractical quantity of material for administration per unit dose of thecompound. Techniques and compositions for making dosage forms useful inthe methods described herein are described in the following references,all incorporated by reference herein: Modern Pharmaceutics, 4th Ed.,Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al.,Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction toPharmaceutical Dosage Forms 8th Edition (2004).

Various oral dosage forms can be used, including such solid forms astablets, capsules, granules and/or bulk powders. Tablets can becompressed, tablet triturates, enteric-coated, sugar-coated,film-coated, or multiple-compressed, containing suitable binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, flow-inducing agents, and/or melting agents. Further soliddosage forms may comprise milled powders, spray-dried powders,crystalline forms, amorphous forms, and glassy forms, which may beadministered as tablets or may be administered as aerosols or airborneparticles, for example for nasal or pulmonary delivery. Liquid oraldosage forms include aqueous solutions, emulsions, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules, and effervescent preparations reconstituted from effervescentgranules, containing suitable solvents, preservatives, emulsifyingagents, suspending agents, diluents, sweeteners, melting agents,coloring agents and/or flavoring agents, or any combination thereof.Further liquid dosage forms may comprise forms for intranasal orpulmonary delivery. Such dosage forms may comprise liquids forintranasal injection, nasal lavage, pulmonary lavage, nebulization oraerosol delivery.

The pharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration in accordance with methodsand compositions of some embodiments herein are well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and/or cellulose; binders such as starch,gelatin and/or sucrose; disintegrants such as starch, alginic acidand/or croscarmellose; lubricants such as magnesium stearate, stearicacid, microcrystalline cellulose, carboxymethyl cellulose, and/or tale.Tablets may also comprise solubilizers or emulsifiers, such aspoloxamers, cremophor/Kolliphor®/Lutrol®, or methylcellulose,hydroxypropylmethylcellulose, or others as are known in the art, or anycombination thereof. Glidants such as silicon dioxide can be used toimprove flow characteristics of the powder mixture. Coloring agents,such as the FD&C dyes, can be added for appearance. Sweeteners andflavoring agents, such as aspartame, saccharin, menthol, peppermint,and/or fruit flavors, or any combination thereof, are useful adjuvantsfor chewable tablets. Capsules typically comprise one or more soliddiluents disclosed above. The selection of carrier components depends onsecondary considerations like taste, cost, and shelf stability, whichcan be readily made by a person skilled in the art.

Peroral (PO) compositions in accordance with methods and compositions ofsome embodiments herein also include liquid solutions, emulsions, orsuspensions. The pharmaceutically-acceptable carriers suitable forpreparation of such compositions are well known in the art. Typicalcomponents of carriers for syrups, elixirs, emulsions and/or suspensionsinclude ethanol, glycerol, propylene glycol, polyethylene glycol, liquidsucrose, sorbitol and/or water. For a suspension, typical suspendingagents include methyl cellulose, sodium carboxymethyl cellulose, AVICELRC-591, tragacanth and/or sodium alginate; typical wetting agentsinclude lecithin and/or polysorbate 80; and typical preservativesinclude methyl paraben and/or sodium benzoate, or any combinationthereof. Peroral liquid compositions may also contain one or morecomponents such as sweeteners, flavoring agents and/or colorants, asdisclosed above.

Such compositions may also be coated by conventional methods, typicallywith pH or time-dependent coatings, such that the subject one or morecompounds are released in the gastrointestinal tract in the vicinity ofthe desired application, or at various times to extend the desiredaction. Exemplary dosage forms for release in the gastrointestinal tractmay incorporate one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethylcellulose, Eudragit coatings, waxes, alginate and/or shellac, or otherexcipients known to those of skill in the art, or any combinationthereof. According to some embodiments, the compositions to beadministered according to the methods described herein are formulatedfor release in the gastrointestinal tract. According to someembodiments, the compositions to be administered according to themethods described herein are formulated for release in the lowergastrointestinal tract. In some embodiments, the compositions areprovided as enteric coated capsules, tablets, soft gels; orintrinsically enteric capsules.

The actual unit dose of the compositions in accordance with methods andcompositions of some embodiments herein depends on the one or morecompounds in the formulation. In some embodiments, the amount of eachcompound in the formulation may be from 0.01 mg/kg to 0.05 mg/kg of bodyweight per day, from 0.04 mg/kg to 0.1 mg/kg of body weight per day,from 0.09 mg/kg to 0.15 mg/kg of body weight per day, from 0.14 mg/kg to0.2 mg/kg of body weight per day, from 0.2 mg/kg to 0.5 mg/kg of bodyweight per day, from 0.4 mg/kg to 1 mg/kg of body weight per day, from 1mg/kg to 6 mg/kg of body weight per day, 5 mg/kg to 500 mg/kg or more ofbody weight per day, from 10 mg/kg or less to 70 mg/kg, from 50 mg/kg to80 mg/kg of body weight per day, from 70 mg/kg to 120 mg/kg of bodyweight per day, from 100 mg/kg to 300 mg/kg of body weight per day, orfrom 250 mg/kg to 500 mg/kg of body weight per day. In some embodiments,the dose may be less than 100 mg/kg, 500 mg/kg, 300 mg/kg, 200 mg/kg,150 mg/kg, 100 mg/kg, 50 mg/kg, 40 mg/kg, 30 mg/kg, 25 mg/kg, 20 mg/kg,10 mg/kg, 7.5 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2.5 mg/kg, or 1mg/kg of body weight per day or an amount that is within a range definedby any two of the aforementioned amounts. In some embodiments, theactual unit dose is 5, 10, 25, 50, 75, 100, 150, or 200 mg/kg of bodyweight per day or an amount that is within a range defined by any two ofthe aforementioned amounts. Thus, for administration to a 70 kg person,for example, the dosage range is from 0.1 mg to 1 mg, 0.9 mtg to 2 mg,from 1.5 mg to 5 mg, from 4 mg to 10 mg, from 9 mg to 20 mg, from 15 mgto 50 mg, from 40 mg to 75 mg, from 50 mg to 100 mg, from 75 mg to 200mg, from 100 mg to 300 mg, from 200 mg to 400 mg, 350 mg to 750 mg, from500 mg to 1 g, from 750 mg to 2 g, from 1 g to 5 g, from 2.5 g to 6 g,from 4 g to 10 g, from 8 g to 20 g, from 15 g to 35 g, or from 1 g orless to 35 g or more, or an amount that is within a range defined by anytwo of the aforementioned amounts. In some embodiments, the actual unitdose is 6 g. In some embodiments, the actual unit dose is 10 g. In someembodiments, the actual unit dose is 35 g. In some embodiments, theactual unit dose is 1 g or less but not zero. In some embodiments, theactual unit dose is 10 g or less but not zero. In some embodiments, theactual unit dose is 35 mg or less but not zero.

“Loading dose,” as used herein refers to an initial dose of a compoundwhich is higher than subsequent doses.

“Maintenance dose.” as used herein refers to a subsequent dose thatfollows a loading dose, and occurs later in time than a loading dose.One of ordinary skill in the art will be aware that the dosage form ormode of administration of a maintenance dose may be different from thatused for the loading dose. In any of the embodiments disclosed herein, amaintenance dose may comprise administration of the unit dosage form onany dosing schedule contemplated herein, including but not limited to,monthly or multiple times per month, biweekly or multiple times each twoweeks, weekly or multiple times per week, daily or multiple times perday. It is contemplated within the present disclosure that dosingholidays may be incorporated into the dosing period of the maintenancedose. Such dosing holidays may occur immediately after theadministration of the loading dose or at any time during the period ofadministration of the maintenance dose. As used herein, the period ofadministration of the maintenance dose may be referred to as the“maintenance phase” of the treatment period.

“Mode of administration” as used herein refers to the avenue by whichone or more compounds are administered to a subject. As used herein,“mode of administration” comprises the dosage form (for example, atablet, powder, dissolved liquid, suspension, emulsion, etc.) andmechanism by which the dosage form is applied to the subject (forexample, by injection, topically, such as by cream, lotion, or patch;orally, such as by a pill, dissolved liquid, oral suspension, buccalfilm, or mouth rinse). As used herein, “mode of administration” alsocomprises the dose, dose amount, and dosing schedule by which a compoundis administered to a subject.

In some embodiments, the compositions to be administered according tothe methods of the present disclosure are provided with, or mixed into,a foodstuff, beverage, or other ingestible item. In some embodiments,said beverage, foodstuff, or other ingestible item may comprise one ormore of a candy, an applesauce, a yogurt, a soft pudding, a gelatinfoodstuff, a juice, milk, a soy or nut beverage, a thickened beverage,or a cheese, or any combination thereof. One of ordinary skill willreadily recognize that the combination of the compositions to beadministered according to the methods of the disclosure can be combinedwith any suitable food or beverage to facilitate ingestion of thecompositions.

In some embodiments in accordance with methods and compositions of someembodiments herein, the mode of administration comprises administering aloading dose followed by a maintenance dose. In some embodiments, theloading dose is 20 g or less but not zero; 15 g or less but not zero; 10g or less but not zero, 6 g or less but not zero, 4 g or less but notzero, 2 g or less but not zero, or 1 g or less but not zero or an amountthat is within a range defined by any two of the aforementioned amounts.In some embodiments, the maintenance dose is 20 g or less but not zero;10 g or less but not zero, 6 g or less but not zero, 4 g or less but notzero, 2 g or less but not zero, 1 g or less but not zero, 500 mg or lessbut not zero, or 250 mg or less but not zero or an amount that is withina range defined by any two of the aforementioned amounts.

In some embodiments in accordance with methods and compositions of someembodiments herein, the loading dose is administered over a period ofone day or 24-hour period. In some embodiments the loading dose isadministered in a single administration. In some embodiments, theloading dose is administered in multiple administrations. In someembodiments, the loading dose is administered in multipleadministrations during a single day or 24-hour period. In someembodiments the loading dose is administered over a period of 2 days. Insome embodiments the loading dose is administered over a period of 3days. In some embodiments the loading dose is administered over a periodof 4 days. In some embodiments the loading dose is administered over aperiod of 5, 6 or 7 days. In some embodiments, the loading dose isadministered over a period of 8-14 days or fewer. In some embodiments,the loading dose is administered over a period of 14 days.

The methods according to the present disclosure contemplate varying orcontrolling the timing of administration of a composition describedherein, in order to enhance the effectiveness of any treatment that isadministered. In some embodiments, a composition to be administeredaccording to the methods of the present disclosure may be administeredwith food, such as concurrently with a meal or other ingestion of afoodstuff. In some further embodiments, a composition to be administeredaccording to the methods of the present disclosure may be administeredimmediately before or immediately after a meal or other ingestion of afoodstuff. In some further embodiments, a composition to be administeredaccording to the methods of the present disclosure may be administeredwithin 1-5 minutes, within 3-10 minutes, within 6-15 minutes, within10-20 minutes, within 15-30 minutes, within 20-45 minutes, or within onehour before or after a meal or other ingestion of a foodstuff. In someembodiments, a composition to be administered according to the methodsof the present disclosure may be administered without food, such asbetween 1-3 hours, between 2-5 hours, between 4-8 hours, between 6-12hours, between 9-18 hours, between 12-24 hours, or more than 24 hoursbefore or after a meal or other ingestion of a foodstuff.

As used herein, “duration of the treatment” refers to the timecommencing with administration of the first dose and concluding with theadministration of the final dose, such length of time being determinedby one of ordinary skill in the art of treating neurological disordersor disorders implicating intestinal hyperpermeability or “leaky gut.”with reference to the symptoms and health of the subject being treatedtherefor. Such duration may be determined with reference to periodic,sporadic, or ongoing monitoring of the levels of amyloid as disclosedherein or as known to one of skill in the art of treating neurologicaldisorders.

As used herein. “dosing holiday” refers to a period of 24 hours or moreduring which either no dose is administered to the subject, or a reduceddose is administered to the subject. As used herein. “reduced dose”refers to a dose that is less than the total daily dose to beadministered to a subject.

According to the present disclosure, the dosing schedule may be variedso as to attain the desired therapeutic effect. In each of theembodiments as disclosed herein, variations in dosing schedule may berepeated throughout the duration of the therapeutic protocol beingadministered. In each of the embodiments as disclosed herein, the firstdosage may be higher, lower, or the same as the dosages following thefirst dosage. In each of the embodiments disclosed herein, a loadingdose may precede the disclosed dosing regimen, and a dosing holiday mayor may not follow the administration of the loading dose.

in some embodiments the methods of the present disclosure compriseadministration of one or more compositions as provided herein daily orless frequently than daily, such as every second day, every third day,every fourth day, every fifth day, every sixth day, or every seventh dayor for a time period that is within a range defined by any two of theaforementioned times. In some embodiments, the compositions as describedherein are formulated for such administration.

According to the methods disclosed herein, a treatment or inhibition ofa disorder implicating amyloid formation may be achieved by modulatingthe dosing schedule for the administration of a composition such thatsubjects experience periodic partial or full reductions in dosing forfixed amounts of time, followed by a resumption of dosing. In someembodiments, dosages are administered daily for between one and thirtydays, followed by a dosing holiday lasting for between one and thirtydays. In some embodiments, during the dosing holiday, no dose isadministered. In some further embodiments, the composition of thepresent disclosure is allowed to clear completely from the subject'sbody prior to administration of the next dose. In some otherembodiments, during the dosing holiday, a dose less than the usual dailydose is administered. In some further embodiments, an amount of theadministered composition less than the therapeutically effective amountis allowed to remain within the subject during the dosing holiday. Insome further embodiments, an amount of the administered compositionsufficient to maintain therapeutic levels in the affected tissues isallowed to remain within the subject. In some embodiments, a compositionis administered at any time following the onset of one or more of theaforementioned symptoms of a neurological disorder associated withamyloid formation. In some embodiments, a composition according to themethods described herein is administered prior to the onset of symptomsof said disorder or disorders. In some embodiments, a compositionaccording to the methods described herein is administered concurrentlywith or after the onset of symptoms of said disorder or disorders.

Methods of Use

The present disclosure provides methods for inhibiting, ameliorating,reducing the likelihood, delaying the onset of, treating, and/orpreventing the amyloid disorder, including methods that inhibit ordisrupt one or more of the following: (1) bacterial amyloid aggregationon the bacterial surface or in the proximal extracellular space; (2) theinteraction between bacterial amyloid and α-synuclein in the GI tract orolfactory system (including enteroendocrine cells and enteric neuronalcells); and/or (3) aggregation of α-synuclein in the GI tract (includingenteroendocrine cells and enteric neuronal cells).

According to the methods of the present disclosure, α-synuclein shouldbe viewed as a representative amyloid protein of the wider range ofknown host amyloid proteins, including one or more of Beta amyloid fromAmyloid precursor protein, Medin, tau. Apolipoprotein AI, Atrialnatriuretic factor, Beta amyloid, Cystatin, IAPP (Amylin), Beta-2microglobulin. Transthyretin, PrP, Gelsolin, Lysozyme, Huntingtin,Keratoepithelin, Calcitonin. Prolactin, Serum amyloid A, superoxidedismutase 1 (SOD1) and/or Immunoglobulin light chain AL, and thecompositions and methods as disclosed herein may be adapted by one ofskill in the art to disrupt the aggregation of any amyloid protein inwhich one amyloid protein (bacterial or human) prompts aggregation ofanother amyloid protein.

Without being limited by theory, representative disorders that presentamyloid formation and the proteins involved in these disorders, whichmay be inhibited or disrupted using the methods of the presentdisclosure, include but are not limited to those disclosed in Table 3.

TABLE 3 Amyloid Disorders Abbre- Disease Protein featured viationAlzheimer's disease (AD) Beta amyloid from Amyloid Aβ, APP precursorprotein Aortic medial amyloid Medin AMed Atherosclerosis ApolipoproteinAI AApoA1 Cardiac arrhythmias, isolated Atrial natriuretic factor AANFatrial amyloidosis Cerebral amyloid angiopathy Beta amyloid Aβ Cerebralamyloid angiopathy Cystatin ACys (Icelandic type) Diabetes mellitus type2 IAPP (Amylin) AIAPP Dialysis related amyloidosis Beta-2 microglobulinAβ2M Familial amyloid Transthyretin ATTR polyneuropathy Fatal familialinsomnia PrP APrP Finnish amyloidosis Gelsolin AGel Hereditarynon-neuropathic Lysozyme ALys systemic amyloidosis Huntington's disease(HD) Huntingtin HTT Lattice corneal dystrophy Keratoepithelin AKerMedullary carcinoma of the Calcitonin ACal thyroid Parkinson's disease(PD) α-synuclein α-Syn Prolactinomas Prolactin APro Rheumatoid arthritis(RA) Serum amyloid A AA Sporadic Inclusion body various, including beta-myositis (S-IBM) amyloid Systemic AL amyloidosis Immunoglobulin light ALchain AL Transmissible spongiform PrP APrP encephalopathy (e.g., bovinespongiform encephalopathy)

The methods of the compositions and methods of the invention can also beused to treat amyloid-mediated disorders of the gastrointestinal tractincluding intestinal dysbiosis, intestinal hyperpermeability, irritablebowel syndrome (IBS), inflammatory bowel disease (IBD), ulcerativecolitis and/or Crohn's disease. These disorders can be associated withone or more symptoms, including dysphagia, reduced gut motility,gastroparesis, constipation (including chronic constipation and chronicidiopathic constipation), small intestinal bacterial overgrowth (SIBO),diarrhea (including chronic diarrhea), abdominal pain and/or cramping,bloating, flatulence, and nausea.

As used herein, the term “intestinal dysbiosis” has its customary andordinary meaning as understood by one of skill in the art in view ofthis disclosure and refers to an imbalance and/or maladaptation of theflora or microbiota within the gut or intestines, and particularly thesmall intestine. Such dysbiosis is characterized by a change in thecomposition of the intestinal or gut microbiome, in terms of thespecies/strains which are present and/or the relative abundance orproportion of the species/strains which are present, in which the changehas a deleterious effect on the host organism. The deleterious effect onthe host organism can result from microbiome-mediated changes inelectrolyte balance, biofilm formation, integrity of the barrier formedby the intestinal epithelial lining, or the release from the microbiomeof metabolites which are directly (e.g., as toxicity or effectors) orindirectly (e.g., as pre-cursors to toxins or effector) injurious to thehealth of the host.

As used herein, the term “intestinal hyperpermeability” has itscustomary and ordinary meaning as understood by one of skill in the artin view of this disclosure. It refers to abnormal increased permeabilityof the barrier formed by the intestinal epithelial lining between theintestinal lumen and the surrounding issues. Such hyperpermeability mayresult from inflammation of the intestinal lining and/or failure of thetight junctions between cells of the intestinal epithelium, which allowsthe passage of substances from the lumen into the surrounding tissueswhere some may enter the peritoneal cavity and/or systemic circulation.Because of this leakage of substances from the gut or intestinal lumen,intestinal hyperpermeability may be referred to as “leaky gut” or “leakygut syndrome.”

As used herein, the term “amyloid disorders.” including variations ofthis root term, includes, but is not limited to any or all of thedisorders of Table 3 as well as amyloid-mediated disorders of thegastrointestinal tract.

As used herein, the term “mammalian amyloid or mammalian amyloidprecursor” includes, but is not limited to, one or more of tau, Betaamyloid from Amyloid precursor protein, Medin, Apolipoprotein AI, Atrialnatriuretic factor, Beta amyloid, Cystatin, IAPP (Amylin), Beta-2microglobulin, Transthyretin, PrP, Gelsolin. Lysozyme, Huntingtin,Keratoepithelin. Calcitonin, Prolactin, Serum amyloid A, and/orImmunoglobulin light chain AL. In certain methods and compositionsdisclosed herein, said microbial amyloid or microbial amyloid precursorcomprises CsgA.

Some embodiments include a method of inhibiting, ameliorating, reducingthe likelihood, delaying the onset of, treating, or preventing anamyloid disorder, the method comprising administering a composition asdescribed herein to a subject in need thereof. The amyloid disorder canbe selected from the group consisting of: α-synucleinopathy, Parkinson'sDisease. Lewy Body Dementia, incidental Lewy body disease. Lewy bodyvariant of Alzheimer's disease, multiple system atrophy, and pureautonomic failure, or any combination of any of these. The amyloiddisorder can also be selected from the group consisting of: intestinaldysbiosis, intestinal hyperpermeability, irritable bowel syndrome (IBS),inflammatory bowel disease (IBD), ulcerative colitis and/or Crohn'sdisease. In some embodiments, the composition administered in the methodcomprises, consists essentially of, or consists of any of the moleculesof Table 1 or Table 2. In some embodiments, the composition administeredin the method comprises, consists essentially of, or consists of acompound of the invention as described herein. In some embodiments ofthe method, the amyloid disorder comprises intestinal amyloidaggregates. For example, the aggregates can comprise a bacterialprotein, for example a curli-associated protein such as CsgA.Accordingly, in some embodiments, the method further comprises detectinga presence or level of such a bacterial protein in an intestinal sampleof the subject, a presence or level of nucleic acids encoding themicrobial (e.g., bacterial) protein, or a presence of level of amicrobial organism that produces the bacterial protein (e.g., acurli-associated protein such as CsgA) in the intestinal sample of thesubject, for example a fecal sample. For example, the protein can bedetected by an immunoassay such as an ELISA, Western Blot, lateral flowassay, no-wash assay or the like. For example, the microbial organismthat produces the microbial protein can be detected by nucleic acidanalysis (such as qualitative or quantitative PCR, microarray analysis,or sequencing). For example, the nucleic acid that encodes the microbialprotein can be detected by qualitative or quantitative PCR, microarrayanalysis, sequencing or branched DNA analysis. An intestinal presence ofthe bacterial protein or microbial organism that produces the protein,or a level of the bacterial protein (or microbial organism that producesthe protein) greater than a control can identify the subject as being inneed of the composition. By way of example, suitable controls caninclude subjects that are negative for the bacterial protein (ormicrobial organisms that make the bacterial protein), for examplehealthy individuals, or an individual identified as not having thebacterial protein (or microbial organisms that make the bacterialprotein) in their intestines. In some embodiments, the method comprisesdetecting a presence or level of intestinal curli (or a curli-associatedprotein such as CsgA), or an intestinal level of a microorganism thatproduces intestinal curli-associated protein (such as CsgA) in a sampleof the subject. In some embodiments, the subject is identified as amember of a subpopulation of subject having the amyloid disorder, and inneed of the composition. In some embodiments, the method furthercomprises determining a decrease or absence of the intestinal amyloidaggregates following the administration.

The compositions of the present disclosure may, in some embodiments,inhibit the formation of α-synuclein aggregates (e.g., fibrils. Lewybodies, or other aggregates) or other host amyloid at its point ofinitiation in the gut, thus depriving microbially induced amyloidaggregation thought to serve as a template or seed for α-synuclein orother host amyloid aggregation and doing so without having to cross theblood brain barrier. Targeting α-synuclein or other host amyloidaggregation in the gut obviates the need for the drug to cross theblood-brain barrier, providing efficacy at a lower dose, with fewerside-effects due to reduction in systemic exposure. Further, targetingα-synuclein or other host amyloid aggregation at its point of initiationallows intervention at an earlier stage in the pathogenic process,preventing or inhibiting disease progression before motor symptoms orother neurodegenerative symptoms develop. Targeting α-synucleinaggregation in the gut may also address gastrointestinal dysfunctionand/or ameliorate gastrointestinal symptoms or behaviors of the subject,which may comprise, e.g., one or more of dysphagia, reduced gutmotility, gastroparesis, constipation (including chronic constipationand chronic idiopathic constipation), small intestine bacterialovergrowth (SIBO), diarrhea, abdominal pain and/or cramping, bloating,flatulence, nausea, or any other symptoms of irritable bowel syndrome(IBS), inflammatory bowel disease (IBD, e.g., ulcerative colitis andCrohn's disease), intestinal hyperpermeability, hypersalivation(sialorrhea), anorectal dysfunction, dyssynergic defecation, or anycombinations thereof, for example in accordance with compositions andmethods of some embodiments herein.

In addition to targeting host amyloid aggregation in the brain as anapproach to treating or inhibiting neurodegenerative diseases, targetingbacterial amyloid aggregation provides new therapies for infectiousdiseases, such as urinary tract infections (UTIs). In both cases,certain classes of compounds have been identified, largely representedas “polyphenols” or polyphenol equivalents, as having the ability toinhibit amyloid aggregation process in tissues of interest, such as inthe brain for α-synuclein and the urinary mucosae for UTIs.

In some embodiments, the compositions and methods of the presentdisclosure contemplate the use of polyphenols and/or polyphenolequivalents as inhibitors of the interaction between a host amyloid,such as α-synuclein and a bacterial amyloid, such as curli or adhesivepili. In some embodiments, the compositions and methods of the presentdisclosure, contemplate the use of polyphenols and/or polyphenolequivalents as inhibitors of host amyloid aggregation and/or promotersof amyloid dis-aggregation in peripheral tissue, such as the gut ornasopharynx, rather than in the brain. The compositions and methods ofthe present disclosure further contemplate modified polyphenols orpolyphenol equivalents, that act locally in the gut and are essentiallynot absorbed into peripheral tissues, such as, for example, non-orallybioavailable analogs of polyphenols that retain amyloid inhibitingactivity, but do not traverse the gut epithelium or enter the primarycirculation.

In some embodiments, the compositions and methods of the presentdisclosure contemplate formulations that enable delivery of saidcompositions to the site of action in the lower small intestine, thelarge intestine, and/or the colon. Said formulations may compriseenteric coated tablets, capsules, liquid-gels or powders, and the like,such that the formulation inhibits the release of the drug in thestomach or upper GI tract. Alternatively, said compositions may compriseintrinsically enteric capsules or similar solid dosage forms wherein thecapsule composition comprises a polymer or material that dissolves at ornear the site of action, such as, for example, EnTrinsic® intrinsicallyenteric capsules, preferably in the lower GI tract, and more especiallythe lower small intestine, the large intestine, or the colon. In someembodiments, said compositions are not absorbed and remain in the GTtract.

The compositions and methods of the present disclosure contemplategut-restricted small molecule inhibitors that target one or moreelements of amyloid formation. Exemplary compounds of the inventioninclude polyphenol moieties, many of which are orally bioavailable. Thecompositions and methods of the present disclosure also contemplategut-selective or gut-restricted, non-orally absorbed derivatives ofnon-polyphenol classes that are known to have the ability to inhibitamyloid formation. The compositions and methods according to the presentdisclosure further contemplate non-orally absorbed, gut-selectivederivatives or formulations of said polyphenol or non-polyphenolcompounds.

“Subject” as used herein, has its customary and ordinary meaning asunderstood by one of skill in the art in view of this disclosure. Itrefers to a human or a non-human mammal including but not limited to adog, cat, horse, donkey, mule, cow, domestic buffalo, camel, llama,alpaca, bison, yak, goat, sheep, pig, elk, deer, domestic antelope, or anon-human primate selected or identified for a diagnosis, treatment,inhibition, amelioration of a neurological disease or neurologicaldisorder associated with microbially induced amyloid, such asParkinson's Disease, Lewy Body Dementia, incidental Lewy body disease,Lewy body variant of Alzheimer's disease, multiple system atrophy, pureautonomic failure, intestinal dysbiosis, intestinal hyperpermeability,irritable bowel syndrome (IBS), inflammatory bowel disease (IBD),ulcerative colitis. Crohn's disease, or any combination thereof.

“Diagnosing” has its customary and ordinary meaning as understood by oneof skill in the art in view of this disclosure. It can refer to the actor process of determining whether a subject exhibits any symptom orindicator of a neurological disease or neurological disorder associatedwith microbially induced amyloid such as Parkinson's Disease. Lewy BodyDementia, incidental Lewy body disease, Lewy body variant of Alzheimer'sdisease, multiple system atrophy, pure autonomic failure, or anycombination thereof. It can also refer to the act or process ofdetermining whether a subject exhibits any symptom or indicator of agastrointestinal disorder associated with microbially induced amyloidsuch as intestinal dysbiosis, intestinal hyperpermeability, irritablebowel syndrome (IBS), inflammatory bowel disease (IBD), ulcerativecolitis and/or Crohn's disease. Diagnosing may further comprise thedetermination of whether the body of a subject or any tissue, fluid,component, organ, or compartment thereof contains microbially inducedamyloid. Diagnosing may further comprise the determination of whetherthe body of a subject or any tissue, fluid, component, organ, orcompartment thereof contains any factor capable of affecting the rate ofaggregation or disaggregation of microbially induced amyloid.

“Subject suspected of having” has its customary and ordinary meaning asunderstood by one of skill in the art in view of this disclosure. Itrefers to a subject exhibiting one or more clinical indicators of adisease or condition. In certain embodiments, the disease or conditionmay comprise one or more of Parkinson's Disease, Lewy Body Dementia,incidental Lewy body disease. Lewy body variant of Alzheimer's disease,multiple system atrophy, pure autonomic failure, or any combinationthereof. In some embodiments, the disorder can be selected from thegroup consisting of: intestinal dysbiosis, intestinal hyperpermeability,irritable bowel syndrome (IBS), inflammatory bowel disease (IBD),ulcerative colitis and/or Crohn's disease.

“Subject in need thereof” has its customary and ordinary meaning asunderstood by one of skill in the art in view of this disclosure. Itrefers to a subject selected or identified as one being in need ofdiagnosis of a disorder implicating amyloid formation, or one in need ofa treatment, inhibition, amelioration of a neurological disease orneurological disorder associated with microbially induced amyloid suchas Parkinson's Disease, Lewy Body Dementia, incidental Lewy bodydisease. Lewy body variant of Alzheimer's disease, multiple systematrophy, pure autonomic failure, or any combination thereof. In otherembodiments, the disorder can be selected from the group consisting of:intestinal dysbiosis, intestinal hyperpermeability, irritable bowelsyndrome (IBS), inflammatory bowel disease (IBD), ulcerative colitisand/or Crohn's disease.

“Microbially induced amyloid” as used herein has its customary andordinary meaning as understood by one of skill in the art in view ofthis disclosure. It refers to amyloid fibrils or aggregates that areproduced through the contact of a mammalian or microbial protein withone or more microbial proteins. Said microbial protein may comprise oneor more proteins of bacterial or fungal origin, although the presentdisclosure contemplates amyloid produced by the interaction of proteins,whatever their origin, with proteins originating from bacteriophages,viruses, bacteria, archaea, fungi, and other eukaryotes.

A “therapeutic effect” has its customary and ordinary meaning asunderstood by one of skill in the art in view of this disclosure. Itrelieves, to some extent, one or more of the symptoms of a disease ordisorder, and includes curing the disease or disorder. “Curing” meansthat the symptoms of active disease are eliminated. However, certainlong-term or permanent effects of the disease may exist even after acure is obtained (such as tissue damage).

“Amelioration” has its customary and ordinary meaning as understood byone of skill in the art in view of this disclosure. It refers to alessening of severity of at least one indicator of a condition ordisease. In certain embodiments, amelioration includes a delay orslowing in the progression of one or more indicators of a condition ordisease. The severity of indicators may be determined by subjective orobjective measures which are known to those skilled in the art.

“Modulation” has its customary and ordinary meaning as understood by oneof skill in the art in view of this disclosure. It refers to analteration in the presence, absolute level, relative level, function oractivity of any factor within the body of a subject or any tissue,fluid, component, organ, or compartment thereof. In certain embodiments,modulation refers to an increase in gene expression. In certainembodiments, modulation refers to a decrease in gene expression. Incertain embodiments, modulation refers to an increase or decrease intotal serum levels of a specific protein. In certain embodiments,modulation refers to an increase or decrease in free serum levels of aspecific protein. In certain embodiments, modulation refers to anincrease or decrease in the aggregation state of a protein. In certainembodiments, modulation refers to increasing or decreasing the stabilityof amyloid fibrils. In certain embodiments modulation refers toincreasing or decreasing the length, width, spacing, or density ofamyloid fibrils. In certain embodiments, modulation refers to anincrease or decrease in total serum levels of a specific non-proteinfactor, e.g., a metabolite. In certain embodiments, modulation refers toan increase or decrease in free serum levels of a specific non-proteinfactor. In certain embodiments, modulation refers to an increase ordecrease in total bioavailability of a specific protein. In certainembodiments, modulation refers to an increase or decrease in totalbioavailability of a specific non-protein factor. In certainembodiments, modulation refers to alterations in the aggregation stateof a protein. In certain embodiments modulation refers to alterations inthe rate or extent of aggregation or disaggregation of microbiallyinduced amyloid.

In some compositions and methods of some embodiments in accordance withthe present disclosure, a subject is selected or identified to receivethe administration of the compositions described herein. In someembodiments, said subject is selected or identified as one havingelevated levels of curli in the gut. Such a selection can be made byclinical or diagnostic evaluation. In some embodiments, said subject isselected or identified as one having elevated levels of microbiallyinduced amyloid in the gut. Such a selection can also be made byclinical or diagnostic evaluation. In some embodiments, said subject isselected or identified as one having elevated levels of α-synuclein inthe gut. Again, such a selection can be made by clinical or diagnosticevaluation. In some further embodiments, said subject is one showing oneor more symptoms of a neurodegenerative disorder, such as ademonstration of anosmia, hyposmia, bradykinesia, ataxia, tremor, musclerigidity, impaired posture and balance, loss of automatic movements,dysarthria or other speech changes, handwriting changes, orthostatichypotension, memory deficit, dysphagia, incontinence, sleep disruption,cardiac arrhythmia, visual disturbance, psychiatric problems includingdepression and visual, auditory, olfactory, or tactile hallucinations,vertigo, cognitive dysfunction, altered dopamine levels, alteredserotonin levels, and/or altered kynurenine levels, gastroparesis,anorectal dysfunction, dyssnergic defecation, or any combinationthereof. In some embodiments, said subject has been diagnosed accordingto methods known in the art of diagnosis of neurological and amyloiddisorders, as having an amyloid disorder. In some further embodiments,said subject has been diagnosed as having or as being at risk of havingLewy Body Dementia, incidental Lewy body disease. Lewy body variant ofAlzheimer's disease, multiple system atrophy, pure autonomic failure, orany combination thereof. In some embodiments, said subject furtherdisplays gastrointestinal symptoms. In some further embodiments, saidgastrointestinal symptoms may comprise one or more of constipation,diarrhea, abdominal pain and/or cramping, bloating, flatulence, nausea,or any other symptoms of irritable bowel syndrome (IBS), inflammatorybowel disease (IBD), ulcerative colitis, Crohn's disease, intestinalhyperpermeability, or any combinations thereof.

In compositions and methods according to some embodiments of the presentdisclosure, a subject selected for treatment may be under the age of 18years. In some embodiments, a subject selected for treatment may bebetween 17 and 30 years of age. In some embodiments, a subject selectedfor treatment may be between 29 and 50 years of age. In someembodiments, a subject selected for treatment may be between 49 and 60years of age. In some embodiments, a subject selected for treatment maybe between 59 and 70 years of age. In some embodiments, a subjectselected for treatment according to the compositions and methodsdescribed herein may be greater than 69 years of age.

In compositions and methods according to some embodiments of the presentdisclosure, administration of one or more of the compositions asdescribed herein provides the effect of preventing the formation of, orpromoting the disaggregation of, amyloid or microbially induced amyloidin the gut, nasal cavity, olfactory bulb, or enteric nervous tissue,e.g., without having to pass the blood brain barrier. In someembodiments, administration of one or more of the compositions asdescribed herein provides the effect of inhibiting the furtheraggregation of amyloid or microbially induced amyloid in the gut, nasalcavity, olfactory bulb, or enteric nervous tissue, e.g., without havingto pass the blood brain barrier. In some embodiments, administration ofone or more of the compositions as described herein provides the effectof causing or enhancing the disaggregation of amyloid or microbiallyinduced amyloid in the gut, nasal cavity, olfactory bulb, or entericnervous tissue, e.g., without having to pass the blood brain barrier. Insome embodiments, administration of one or more of the compositions asdescribed herein provides the effect of causing or enhancing thedisaggregation of preexisting amyloid or microbially induced amyloid inthe gut, nasal cavity, olfactory bulb, or enteric nervous tissue, e.g.,without having to pass the blood brain barrier. In some embodiments,administration of one or more of the compositions as described hereinprovides the effect of preventing the development of one or moresymptoms of one or more neurological or neurodegenerative disorders. Insome embodiments, administration of one or more of the compositions asdescribed herein provides the effect of ameliorating one or moresymptoms of one or more neurological or neurodegenerative disorders. Insome embodiments, administration of one or more of the compositions asdescribed herein provides the effect of reversing one or more symptomsof one or more neurological or neurodegenarative disorders. In someembodiments, said one or more symptoms of one or more neurologicaldisorders may comprise one or more of anosmia, hyposmia, bradykinesia,ataxia, tremor, muscle rigidity, impaired posture and balance, loss ofautomatic movements, dysarthria or other speech changes, handwritingchanges, orthostatic hypotension, memory deficit, dysphagia,incontinence, sleep disruption, cardiac arrhythmia, visual disturbance,psychiatric problems including depression and visual, auditory,olfactory, or tactile hallucinations, vertigo, cognitive dysfunction,altered dopamine levels, altered serotonin levels, and/or alteredkynurenine levels, gastroparesis, anorectal dysfunction, dyssnergicdefecation, or any combination thereof. In some embodiments, said one ormore neurological disorders may comprise an amyloid disorder. In somefurther embodiments, said one or more neurological disorders maycomprise one or more of Lewy Body Dementia, incidental Lewy bodydisease, Lewy body variant of Alzheimer's disease, multiple systematrophy, and/or pure autonomic failure, or any combination thereof.

In other embodiments, the inhibitors of amyloid formation may beintended for administration systemically or locally to the enteric ofcentral nervous system. For example, inhibitors which are effectiveagainst mammalian amyloid or mammalian amyloid precursor proteinaggregation may be useful in treatment of one or more of the amyloiddisorders described herein (Table 3). Therefore, for such embodiments,the compositions comprising the inhibitors of amyloid formation may beformulated for parenteral administration, including systemicadministration (e.g., intravenous, subcutaneous, intramuscular,intraperitoneal) or local administration (e.g., local injection near thevagus nerve, intraspinal injection, or intracranial injection). Fordelivery into the CNS, it is necessary for the inhibitors to passthrough the blood brain barrier. Therefore, in such embodiments, theinhibitors are preferably lipid soluble molecules, or may be modified toincrease lipid solubility, or may be co-administered with compounds thatenhance passage through the blood brain barrier (see, e.g.,WO2014076655A1, WO2012159052A2, WO1992018529A1).

In compositions and methods according to some embodiments of the presentdisclosure, levels of amyloid and/or microbially induced amyloid in thetissues, fluids, or feces of the subject are monitored or evaluatedduring the course of therapy. In some further embodiments, levels ofamyloid and/or microbially induced amyloid are monitored before and/orafter the course of therapy. In some embodiments, levels of α-synucleinin the tissues, fluids, or feces of the subject are monitored during thecourse of therapy. In some embodiments, levels of α-synuclein aremonitored before and/or after the course of therapy. In someembodiments, measurement of amyloid, microbially induced amyloid, and/orα-synuclein are measured in a fecal sample from the subject. In someembodiments, measurement of amyloid, microbially induced amyloid, and/orα-synuclein are measured in a tissue sample from the subject. In someembodiments, said tissue sample comprises gut epithelium, peritoneum,enteric nervous tissue, olfactory tissue, nasal endothelium, sinusendothelium, brain, and/or nervous tissue. In some embodiments, saidtissue sample comprises cerebrospinal fluid or synovial fluid. In someembodiments, said tissue sample comprises blood, lymph, or plasma.

Methods to Identify Compounds

Disclosed herein are methods to identify compounds, which alter theability of bacterial amyloid to promote aggregation and amyloidformation of the eukaryotic protein α-synuclein. Further disclosedherein are methods of screening for entities useful for the treatment orinhibition of neurodegenerative diseases and screening for entitiesuseful for the prevention or amelioration of the progression ofneurodegenerative diseases. Further disclosed herein are methods ofscreening for entities useful for the treatment or inhibition ofgastrointestinal dysfunction related to neurodegenerative diseases.Additionally disclosed herein are methods for studying the molecularetiology of mammalian amyloid diseases and the molecular link betweenbacterial amyloid production and mammalian amyloid production. Accordingto the methods of the present disclosure, said neurodegenerativediseases and/or mammalian amyloid diseases may comprise one or more ofParkinson's disease (PD), Lewy body dementia, multiple system atrophy,and all other α-synucleinopathies, PD-associated constipation.PD-associated hyposmia, Huntington's Disease, Alexander's Disease,amyotrophic lateral sclerosis (ALS), Alzheimer's Disease and otherdiseases in which amyloids are implicated.

The methods as disclosed herein comprise a suite of in vitro assays thatmeasure one or more of the following (1) bacterial amyloid aggregationon the bacterial surface or in the proximal extracellular space; (2) theinteraction between bacterial amyloid and α-synuclein in the GT tract orolfactory system (including enteroendocrine cells and enteric neuronalcells); or (3) aggregation of α-synuclein in the GI tract (includingenteroendocrine cells and enteric neuronal cells). According to themethods of the present disclosure, α-synuclein should be viewed as arepresentative amyloid protein of the wider range of known mammalianamyloid or mammalian amyloid precursor proteins, and the methods asdisclosed herein may be adapted by one of skill in the art to evaluatethe aggregation of any amyloid protein in which a one amyloid protein(bacterial or human) prompts aggregation of another amyloid protein.Representative disorders that present amyloid formation and the proteinsinvolved in these disorders, which may be evaluated using the methods ofthe present disclosure, include but are not limited to those disclosedin Table 3. Accordingly, in some embodiments, the methods comprisecontacting a plurality of concentrations of a microbial amyloid or amicrobial amyloid precursor with a plurality of concentrations ofα-Synuclein and/or other mammalian amyloid or mammalian amyloidprecursor in the presence of a composition, analyzing or measuring theformation or disaggregation of amyloid after the reaction set forthabove; and comparing said analysis or measurement to an analysis ormeasurement of a control, wherein said control comprises analyzing ormeasuring the formation of amyloid after the reaction set forth above inthe absence of said composition. In certain methods and compositionsdisclosed herein, said microbial amyloid or microbial amyloid precursorcomprises CsgA.

In some embodiments, the methods according to the present disclosurecontemplate contacting a microbial amyloid or a microbial amyloidprecursor (e.g., a composition comprising CsgA) with varyingconcentrations of a mammalian amyloid or mammalian amyloid precursor inthe presence of a composition, said composition comprising a compound ormixture to be tested for its ability to inhibit amyloid formation orenhance amyloid disaggregation. In some further embodiments, saidcombination of microbial amyloid or microbial amyloid precursor,mammalian amyloid or mammalian amyloid precursor, and test compositionare analyzed or measured for changes in the amount of amyloid present.In some further embodiments, the rate and/or extent of amyloid formationwithin said combination of microbial amyloid or microbial amyloidprecursor, mammalian amyloid or mammalian amyloid precursor, and testcomposition is compared to the rate of amyloid formation within acontrol sample lacking said composition. In some embodiments, the rateof formation of amyloid is measured. In some further embodiments, thetotal amount of amyloid formation is measured. In some furtherembodiments, the temperature of the assay is varied, whereby thestability of the newly-formed amyloid fibrils is measured relative tothose formed under native conditions. In some embodiments, the methodsare carried out by placing said composition within the wells of amulti-well assay plate. In some further embodiments, the methodsaccording to the present disclosure are carried out in the presence of aphysical agitator. In some further embodiments, said physical agitatorcomprises glass, teflon, or polymer beads. In some further embodiments,said polymer beads may comprise polystyrene, polylactic acid, polylactic-co-glycolic acid, polycarbonate, or polytetrafluoroethylene(Teflon®) beads. In some embodiments, the beads or objects used foragitation will be from 10-1000 μm in their longest dimension. In someembodiments, the beads or objects used for agitation are from 10-100 μm,from 80-200, from 180-300 μm, from 280-400 μm, from 380-500 μm, from480-600 μm, from 580-700 μm, from 680-800 μm, from 780-900 μm, or from880-1000 μm in their longest dimension. In some embodiments, the beadsor objects used for agitation will be greater than 1 mm in their longestdimension. In some embodiments, the beads or objects used for agitationwill be less than 10 mm in their longest dimension. In certainembodiments, the beads or objects are 1-3 mm, 1-5 mm, 2-5 mm, 3-5 mm,4-5 mm, 5-6 mm, 5-7 mm, 5-8 mm, 5-9 mm, 5-10 mm, 2-10 mm, 4-10 mm, 6-10mm, or 8-10 mm. In certain particular embodiments, the beads or objectsare 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm intheir longest dimension.

In some embodiments, the microbial amyloid or microbial amyloidprecursor comprises CsgA, the major protein constituent of curli, alsoknown as adhesive pili, or any analogue or homologue thereof. In someembodiments, the microbial amyloid or microbial amyloid precursorcomprises CsgB, which nucleates the conversion of CsgA to its amyloidform, or polypeptides derived therefrom. In some embodiments, saidmammalian amyloid or mammalian amyloid precursor comprises α-synuclein.

In some embodiments, contacting microbial amyloid or a microbial amyloidprecursor (e.g., a composition comprising CsgA) with varyingconcentrations of mammalian amyloid or mammalian amyloid precursor witha composition occurs in the presence of an indicator of amyloidformation. In some further embodiments, said indicator may comprise afluorescent indicator, in which the fluorescence intensity of theindicator varies in a manner correlated with the amount of amyloidpresent in the sample. Said variation may occur due to changes influorescence related to changes in the molecular environment associatedwith interposition of the label into the assembling amyloid fibril. Insome further embodiments, said indicator may comprise thioflavin T(ThT). In some embodiments, labels that are bound to amyloid precursormolecules may show changes in intensity or wavelength of emission due tointermolecular fluorescence quenching or fluorescence resonance energytransfer that is correlated with the formation of amyloid fibrils.Exemplary fluorescent labels are disclosed in The Molecular ProbesHandbook (Invitrogen. Inc., 2010), which is hereby incorporated byreference for its teachings regarding FRET pairs, fluorescencequenching, and fluorescent probes conjugatable to proteins. Otherexemplary fluorescent labels may comprise fluorescence proteins,including but not limited to the Green fluorescent protein (GFP), theYellow Fluorescent Protein (YFP), AmCyan1, AsREd2, mBanana, mCherry,Dendra2, DsRed2, DsRed-express, DsRed-monomer, DsRed, E2-Crimson,GFP-UV, the Blue Fluorescent Protein (BFP), HcRed1, mOrange, PAmCherry,mPlum, mRaspberry, mStrawberry, tdTomato, ZsGreen1, ZsYellow1, orAcGFP1, or their derivatives, or others fluorescent proteins as areknown in the art. In some further embodiments, the label attached to themammalian amyloid precursor is different from the label that is attachedto the bacterial amyloid or bacterial amyloid precursor. In someembodiments, the bacterial amyloid or bacterial amyloid precursor isunlabeled. In some embodiments, the mammalian amyloid, mammalian amyloidprecursor, bacterial amyloid precursor, or bacterial amyloid containmore than one label. In some further embodiments, said indicator maycomprise a colorimetric indicator, a spin label (such as, for example,3H, 15N or 13C), a metal ion binding compound (such as, for example, aporphyrin, chelator, polyhistidine, or other metal binding polypeptide),an enzyme, or an amyloid-specific antibody. In some embodiments, thedevelopment of amyloid fibrils is observed directly by opticalmicroscopy. In some embodiments, amyloid formation is observed by directlight transmission, or by reflectivity. In some embodiments, amyloidformation is observed by total internal reflection FTIR. In someembodiments, amyloid formation is observed by NMR, FTIR, SPIR, or SPRspectroscopy. In some embodiments, amyloid formation is observed and/orconfirmed by optical birefringence. In some embodiments, samples arestained with congo red dye prior to visualization. In some embodiments,amyloid formation is observed by Raman scattering. In some embodiments,amyloid formation is observed by monitoring changes in the internalfluorescence of the sample, such as that due to internal tryptophan,tyrosine, phenylalanine, histidine, and arginine residues. In someembodiments, amyloid formation is observed by monitoring the binding ofan amyloid-specific antibody, by means as are known in the art such asby conjugation of said antibody to a fluorescent label, a colorimetriclabel, a spin label, a radioisotope, and enzyme, a fluorescent protein,a metal binding domain or other methods known to those of ordinary skillin the art for the detection or visualization of antibodies. Accordingto the methods as described herein, said antibody may comprise anantibody with binding activity that is selective for either amyloid, oramyloid precursor.

In some embodiments, the methods of the present disclosure may becarried out by monitoring the kinetics of fluorescence intensity of anamyloid specific dye in the presence of a mammalian amyloid precursor,and one or more bacterial amyloid precursors or aggregates. In someembodiments, said mammalian amyloid precursor is α-synuclein. In someembodiments, said bacterial amyloid precursor or aggregate is CsgA. Insome embodiments, said amyloid specific dye is Thioflavin T.

In some embodiments, the present disclosure contemplates a kit for thepractice of the methods described herein. In some embodiments, said kitcomprises at least a mammalian amyloid or mammalian amyloid precursor, abacterial amyloid or bacterial amyloid precursor, an indicator ofamyloid formation as described herein, wherein such indicator may or maynot be conjugated to said mammalian amyloid or mammalian amyloidprecursor, a bacterial amyloid or bacterial amyloid precursor, and oneor more reaction vessels. Said kit may comprise a multi-well plate. Saidkit may further comprise instructions for the carrying out of themethods described herein.

The methods of the present disclosure provide methods of screeningcandidate compounds in order to identify compounds that modulate theaggregation and/or disaggregation of amyloid, especially microbiallyinduced amyloid. In some embodiments, the methods of the presentdisclosure comprise the screening of a library of candidate compounds.In some further embodiments, the compositions contacted with mammalianamyloid or mammalian amyloid precursor, and bacterial amyloid precursoror bacterial amyloid, according to the methods disclosed herein,comprise one or more compounds, or combinations thereof, suspected inthe art to inhibit amyloid formation or to destabilize or disaggregateexisting amyloid. In certain embodiments the compositions contacted withmammalian amyloid or mammalian amyloid precursor, and bacterial amyloidprecursor or bacterial amyloid, according to the methods disclosedherein, comprise a natural product or an extract from a natural product.In some embodiments the compositions contacted with mammalian amyloid ormammalian amyloid precursor, and bacterial amyloid precursor orbacterial amyloid, according to the methods disclosed herein, comprisean herb, herbal extract, or botanical substance. In some embodiments,said compositions may comprise tissue or fluid from an animal, plant, orfungus. In some further embodiments, said compositions may comprisetissue, fluid, or extracts of tissue or fluid, from a seed, fruit,flower, leaf, stem, cambium, or root of a plant, or combinationsthereof. In some further embodiments, said compositions may comprisetissue, fluid, or extracts of a tissue or fluid, from the feces, urine,blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, orany internal organ of an animal. In some embodiments, said compositionmay comprise one or more bacteria, or lysates, extracts, conditionedculture media, lyophilized bacteria, lyophilized lysates, lyophilizedculture media thereof, or any combination thereof. In some embodimentssaid bacteria comprise one or more of Bacteroides, Prevotella,Parabacteroides, Faecalibacterium, Eubacterium, Roseburia, Blautia,Coprococcus, and Bifidobacterium, or any combination thereof.

In some embodiments, the methods of the present disclosure can be usedto diagnose or assess the risk for developing an amyloid disorder in asubject. The methods of the present disclosure may be used in thetreatment, prevention, and/or amelioration of one or more neurologicaldisorders including Parkinson's Disease, Lewy Body Dementia, incidentalLewy body disease. Lewy body variant of Alzheimer's disease, multiplesystem atrophy, pure autonomic failure, or any combination thereof. Thedisorders may include behavioral symptoms as are known in the art ofclinical diagnosis and treatment of neurological disorders such ascommunicative symptoms, stereotyped behaviors, sensorimotor issues,and/or anxiety-like behaviors in addition to physical symptoms as areknown in the art of diagnosis and treatment of neurological disorderssuch as tremors, paralysis, dyskinesia, and/or gastrointestinal symptomssuch one or more of constipation, diarrhea, abdominal pain and/orcramping, bloating, flatulence, nausea, or any other symptoms ofirritable bowel syndrome (IBS), inflammatory bowel disease (IBD),ulcerative colitis, Crohn's disease, intestinal hyperpermeability, orany combinations thereof. Accordingly, such clinical and/or diagnosticevaluations and determinations can be used to identify and/or select oneor more subjects for receiving one or more compounds described herein inaccordance with the one or more methods provided in this disclosure. Themethods of the present disclosure may, in some embodiments, includemonitoring of the behavioral, physical, and/or gastrointestinal symptomsas are known in the art of diagnosis and treatment of neurologicaldisorders. In some embodiments, the methods according to the presentdisclosure incorporate monitoring changes in the behavior of a subject.In some further embodiments, the methods according to the presentdisclosure incorporate monitoring the subject for behavioral symptoms asare known to be related to Parkinson's Disease. Lewy Body Dementia,incidental Lewy body disease, Lewy body variant of Alzheimer's disease,multiple system atrophy, pure autonomic failure, or any combinationthereof. In some further embodiments, the methods according to thepresent disclosure incorporate monitoring the subject for bradykinesia,ataxia, tremor, muscle rigidity, impaired posture and balance, loss ofautomatic movements, dysarthria or other speech changes, handwritingchanges, orthostatic hypotension, memory deficit, dysphagia,incontinence, sleep disruption, cardiac arrhythmia, visual disturbance,psychiatric problems including depression and visual, auditory,olfactory, or tactile hallucinations, vertigo, cognitive dysfunction, orany combination thereof or any other symptom known to those in the artof neurological diagnosis or treatment to be useful in the diagnosis ofamyloid disorders, and especially α-synucleinopathies. In some furtherembodiments, the methods according to the present disclosure incorporatemonitoring the subject for gut motility, including gastroparesis,colonic motility, anorectal dysfunction and dyssynergic defecation.Again, such clinical and/or diagnostic evaluations and determinationscan be used to identify and/or select one or more subjects for diagnosisand/or treatment according to the methods described herein. In someembodiments, the methods of the present disclosure may includemonitoring of levels of bacterial, host-derived, and microbially-inducedamyloid as disclosed herein in addition to the aforementioned clinicalmonitoring. According to the methods of the present disclosure, saidamyloid may be monitored in the gut, feces, urine, blood, saliva,cerebrospinal fluid, and/or synovial fluid of a subject. The methods ofthe present disclosure contemplate the monitoring of said amyloid in anytissue or fluid obtainable from a subject during the course oftreatment, and thereby identifying whether said sample contains factorswhich enhance or inhibit amyloid formation. In some embodiments, asubject from whom a tissue, fluid, or other sample is derived, for whichsample the assays described herein indicate the presence of factors,which enhance or accelerate amyloid formation, may be considered to beat elevated risk of developing an amyloid disorder. In some embodiments,said subject may be administered a drug or treatment to ameliorate orprevent said amyloid disorder. Again, such clinical and/or diagnosticevaluations and determinations can be used to identify and/or select oneor more subjects for receiving one or more compounds described herein inaccordance with the one or more methods provided in this disclosure.

According to the methods disclosed herein, a treatment or inhibition ofa disorder implicating amyloid formation may be achieved by modulatingthe dosing schedule for the administration of a composition such thatsubjects experience periodic partial or full reductions in dosing forfixed amounts of time, followed by a resumption of dosing. In someembodiments, dosages are administered daily for between one and thirtydays, followed by a dosing holiday lasting for between one and thirtydays. In some embodiments, during the dosing holiday, no dose isadministered. In some further embodiments, the composition of thepresent disclosure is allowed to clear completely from the subject'sbody prior to administration of the next dose. In some otherembodiments, during the dosing holiday, a dose less than the usual dailydose is administered. In some further embodiments, an amount of theadministered composition less than the therapeutically effective amountis allowed to remain within the subject during the dosing holiday. Insome further embodiments, an amount of the administered compositionsufficient to maintain therapeutic levels in the affected tissues isallowed to remain within the subject. In some embodiments, a compositionis administered at any time following the onset of one or more of theaforementioned symptoms of a neurological disorder associated withamyloid formation. In some embodiments, a composition according to themethods described herein is administered prior to the onset of symptomsof said disorder or disorders. In some embodiments, a compositionaccording to the methods described herein is administered concurrentlywith or after the onset of symptoms of said disorder or disorders.

The following items are set forth in accordance with some embodimentsherein.

1. A method of disrupting and/or inhibiting the formation of amyloidaggregates comprising contacting amyloid or a precursor of amyloid witha composition comprising a compound of the invention.

2. A method of inhibiting the formation of amyloid aggregates comprisingcontacting amyloid or a precursor of amyloid with a compositioncomprising a compound of the invention.

3. A method of disrupting the formation of amyloid aggregates in asubject comprising:

administering to said subject a composition comprising a compound of theinvention; andoptionally, selecting said subject to receive the benefit of a moleculethat disrupts the formation of amyloid aggregates, such as by clinicalor diagnostic evaluation, prior to administering said composition;and/oroptionally, measuring a disruption or inhibition of the formation ofamyloid aggregates in said subject after administration of saidcomposition.

4. A method of disrupting the formation of amyloid aggregates comprisingcontacting amyloid or a precursor of amyloid with a compositioncomprising a compound of the invention.

5. A method of inhibiting, ameliorating, reducing the likelihood,delaying the onset of, treating, or preventing an amyloid disorder, themethod comprising administering to a subject in need thereof a compoundof the invention, or a pharmaceutical composition thereof.

6. The method of item 5, wherein the amyloid disorder is selected fromthe group consisting of: α-synucleinopathy. Parkinson's Disease, LewyBody Dementia, incidental Lewy body disease, Lewy body variant ofAlzheimer's disease, multiple system atrophy, or pure autonomic failure,or any combination thereof.

7. The method of any one of Items 5-6, wherein the amyloid disordercomprises intestinal amyloid aggregates, for example aggregates thatcomprise a bacterial protein such as CsgA.

8. The method of any one of Items 5-7, wherein the amyloid disorder isintestinal dysbiosis, intestinal hyperpermeability, irritable bowelsyndrome (IBS), inflammatory bowel disease (IBD), ulcerative colitis andCrohn's disease.

9. The method of any one of Items 5-8, further comprising detecting apresence or level of a bacterial protein, such as CsgA, nucleic acidsencoding the microbial protein, or a microorganism that produces thebacterial protein in an intestinal sample of the subject.

10. The method of Item 9, wherein the subject is selected as in need ofthe composition if a presence of the bacterial protein or themicroorganism that produces the bacterial protein is detected in theintestinal sample, or if a level of the bacterial protein or themicroorganism that produces the bacterial protein in the intestinalsample is greater than a predetermined level or control.

11. The method of any one of Items 7-10, further comprising determininga decrease or absence of the intestinal amyloid aggregates following theadministration.

12. The method of any one of Items 7-11, further comprising identifyingthe subject as displaying a gastrointestinal symptom.

14. The method of any one of Items 5-12, wherein the subject suffersfrom gastrointestinal symptoms comprising one or more of dysphagia,reduced gut motility, gastroparesis, constipation (including chronicconstipation and chronic idiopathic constipation), small intestinebacterial overgrowth (SIBO), diarrhea, abdominal pain and/or cramping,bloating, flatulence, hypersalivation (sialorrhea), anorectaldysfunction, dyssynergic defecation, and nausea.

16. The method of any of Items 1-14, wherein said composition isformulated for enteric or intranasal delivery.

17. The method of any of Items 1-16, wherein said composition isformulated for controlled release within the lower intestine or colon.

18. The method of any of Items 1-17, wherein said composition is anenteric-coated capsule, tablet, soft-gel, spray dried powder, polymermatrix, hydrogel, enteric-coated solid, crystalline solid, amorphoussolid, glassy solid, coated micronized particle, liquid, nebulizedliquid, aerosol, or microcapsule.

19. The method of any of Items 1-18, wherein said amyloid aggregatescomprise one or more mammalian proteins such as, any one or more ofα-synuclein, tau, Beta amyloid from Amyloid precursor protein, Medin,Apolipoprotein AI, Atrial natriuretic factor, Beta amyloid, Cystatin,IAPP (Amylin), Beta-2 microglobulin. Transthyretin, PrP, Gelsolin,Lysozyme, Huntingtin, Keratoepithelin, Calcitonin, Prolactin, Serumamyloid A, SOD1, and/or Immunoglobulin light chain AL.

20. The method of any of Items 1-19, wherein said amyloid aggregatescomprise one or more bacterial or fungal proteins, such as CsgA.

21. The method of any of Items 1-20, wherein said amyloid aggregatescomprise a bacterial protein, such as CsgA.

22. The method of any of Items 1-21, wherein said amyloid aggregates arepresent within the gastrointestinal tract, cranial sinus, or nasalcavity.

23. The method of any of Items 1-22, wherein said amyloid aggregates arepresent within enteric nervous tissue or the olfactory bulb.

24. The method of any of Items 1-23, wherein the composition isadministered daily.

25. The method of any of Items 1-24, wherein the composition isadministered multiple times per day.

26. The method of any of Items 1-25, wherein the composition isadministered less frequently than daily.

27. The method of any of Items 1-24 or 26, wherein the composition isadministered every second day, every third day, every fourth day, everyfifth day, every sixth day, or every seventh day.

28. The method of any of items 1-27, further comprising measuring orevaluating enteric amyloid levels and/or amyloid aggregation during thecourse of administration.

29. The method of any of Items 1-28, further comprising measuring orevaluating enteric amyloid levels and/or amyloid aggregation followingthe course of administration.

30. The method of any of Items 1-29, further comprising measuring orevaluating a change in the nervous system, such as a neurologicalsymptom or behavior of the subject.

31. The method of any of Items 1-30, wherein said subject is under theage of 18, 18-30, 30-50, 50-60, 60-70, or over the age of 70.

32. The method of any of Items 1-31, further comprising measuring orevaluating a change in the gastrointestinal system, such as agastrointestinal symptom or behavior of the subject.

33. The method of Item 32, wherein said gastrointestinal symptomcomprises constipation.

34. The method of any of Items 1-33, wherein said subject suffers fromgastrointestinal symptoms comprising one or more of constipation,diarrhea, abdominal pain and/or cramping, bloating, flatulence, nausea,or any other symptoms of irritable bowel syndrome (IBS), inflammatorybowel disease (IBD, such as ulcerative colitis and Crohn disease),intestinal hyperpermeability, or any combinations thereof.

35. The method of any of Items 1-34, wherein the composition isadministered following the appearance of a neurological symptom orcondition.

36. The method of Item 35, wherein said neurological symptom orcondition comprises one or more of anosmia, hyposmia, bradykinesia,ataxia, tremor, muscle rigidity, impaired posture and balance, loss ofautomatic movements, dysarthria or other speech changes, handwritingchanges, orthostatic hypotension, memory deficit, dysphagia,incontinence, sleep disruption, cardiac arrhythmia, visual disturbance,psychiatric problems including depression and visual, auditory,olfactory, or tactile hallucinations, vertigo, cognitive dysfunction,altered dopamine levels, altered serotonin levels, altered kynureninelevels, and/or any combination thereof.

37. The method of any of Items 1-36, wherein the composition isadministered prior to the appearance of a neurological symptom orcondition.

38. The method of any of Items 1-37, wherein the method is repeated.

39. The method of any of Items 1-38, wherein, for a givenadministration, the composition is different from a compositionpreviously administered.

40. The method of any of Items 1-39, wherein, for a givenadministration, the dose administered is different from a dosagepreviously administered.

41. The method of any of Items 1-40, wherein the composition isco-administered with a caffeine, nicotine, theophylline, theobromine,xanthine, methylxanthine, or derivatives thereof.

42. The method of any of Items 1-41, further comprising administering tosaid subject an inhibitor of α-synuclein aggregation.

43. The method of any of Items 1-42, wherein said subject is one thathas been identified or selected as being at risk for developing oralready having Parkinson's disease, such as by clinical or diagnosticevaluation.

44. The method of any of Items 1-43, wherein said subject is one thathas been identified or selected as being at risk for developing oralready having Lewy Body Dementia, incidental Lewy body disease. Lewybody variant of Alzheimer's disease, multiple system atrophy, pureautonomic failure, or any combination thereof, such as by clinical ordiagnostic evaluation.

45. The method of item 14, wherein the gastrointestinal symptoms areassociated with Parkinson's Disease or Parkinsonism.

46. The method of any one of items 1-44, wherein the amyloid disordercan be diagnosed by detecting the presence or level of intestinalbacterial amyloid aggregates.

Additional Options

The following options are set forth in accordance with some embodimentsherein.

1. A method of identifying a composition that affects the formation ofmicrobially-induced amyloid, comprising:

-   -   (a) contacting a plurality of concentrations of a microbial        amyloid or a microbial amyloid precursor (e.g., CsgA) with a        plurality of concentrations of α-Synuclein in the presence of a        composition comprising a compound of the invention;    -   (b) analyzing or measuring the formation of amyloid produced by        the reaction set forth in (a); and    -   (c) comparing the analysis or measurement made in (b) with an        analysis or measurement of a control, wherein said control        comprises analyzing or measuring the formation of amyloid after        the reaction set forth in (a) in the absence of said        composition.

2. The method of Option 1, wherein said microbial amyloid or microbialamyloid precursor comprises CsgA.

3. The method of Options 1 or 2, further comprising agitation during(a).

4. The method of Options 1-3, wherein the contacting performed in (a) isconducted in the presence of an indicator of amyloid formation.

5. The method of Option 4, wherein said indicator is a fluorescentindicator, a spin-labeled indicator, an enzyme, an antibody, or acolorimetric indicator.

6. The method of Option 4, wherein said indicator is Thioflavin T.

7. The method of Option 4 wherein said antibody has specificity foraggregated α-Synuclein, and wherein said antibody optionally isconjugated to a fluorescent label, an enzyme, a colorimetric label, aspin label, a metal ion binding moiety, a nucleic acid, apolysaccharide, or a polypeptide.

8. The method of any of Options 1-7, wherein said CsgA and saidα-Synuclein are each separately labeled.

9. The method of any of Options 1-8, wherein the formation is analyzedor measured by internal fluorescence, by fluorescence of a dye or label,by fluorescence resonance energy transfer, by fluorescence polarization,by fluorescence polarization transfer, by UV/Vis Spectroscopy, bymagnetic resonance, by Raman scattering, by electron paramagnetic spinresonance, by light microscopy, by electron microscopy, by scanningtunneling microscopy, or by atomic force microscopy.

10. The method of any of Options 1-9, wherein said composition comprisesa mixture of compounds.

11. The method of any of Options 1-10, wherein said compositioncomprises tissue, bodily fluid or an extract thereof.

12. The method of any of Options 1-11, wherein said compositioncomprises feces, urine, blood, spinal fluid, or saliva, or a componentthereof.

16. The method of any of Options 1-10 wherein said composition comprisesone or more bacteria, bacterial extracts, lysates, conditioned culturemedia, lyophilized bacteria, lyophilized lysates, lyophilized culturemedia, or any combination thereof.

17. The method of any of Options 1-16, further comprising identifying orselecting compositions that alter amyloid formation.

18. The method of any of Options 1-17, further comprising identifying orselecting compositions that reduce amyloid formation.

19. The method of any of Options 1-18, wherein the rate of formation ofamyloid is analyzed or measured in (b).

20. A method of making microbially-induced amyloid, comprising:

-   -   (a) contacting a plurality of concentrations of a microbial        amyloid or a microbial amyloid precursor (e.g., CsgA) with a        plurality of concentrations of α-Synuclein in the presence of a        composition comprising a compound of the invention;    -   (b) providing conditions that allow for the formation of new        microbially-induced amyloid; and    -   (c) analyzing or quantifying the microbially-induced amyloid        formed in (b).

21. The method of Option 20, wherein said microbial amyloid or microbialamyloid precursor comprises CsgA.

22. The method of Options 20 or 21, further comprising agitation during(a).

23. The method of any of Options 20-22, wherein the contacting performedin (a) is conducted in the presence of an indicator of amyloidformation.

24. The method of Option 23, wherein said indicator is a fluorescentindicator, a spin-labeled indicator, or a colorimetric indicator.

25. The method of Options 23 or 24, wherein said indicator is ThioflavinT.

26. The method of any of Options 20-25, wherein said CsgA and saidα-Synuclein are each separately labeled.

27. The method of any of Options 20-26, wherein the formation isanalyzed or measured by internal fluorescence, by fluorescence of a dyeor label, by fluorescence resonance energy transfer, by fluorescencepolarization, by fluorescence polarization transfer, by UV/VisSpectroscopy, by magnetic resonance, by Raman scattering, by electronparamagnetic spin resonance, by light microscopy, by electronmicroscopy, by scanning tunneling microscopy, or by atomic forcemicroscopy.

28. The method of any of Options 20-27, wherein said compositioncomprises a mixture of compounds.

29. The method of any of Options 20-28, wherein said compositioncomprises tissue, bodily fluid or an extract thereof.

30. The method of any of Options 20-29, wherein said compositioncomprises feces, urine, blood, spinal fluid, or saliva, or a componentthereof.

31. The method of any of Options 28-30 wherein said compositioncomprises one or more bacteria, bacterial extracts, lysates, conditionedculture media, lyophilized bacteria, lyophilized lysates, lyophilizedculture media, or any combination thereof.

35. The method of any of Options 20-34, further comprising identifyingor selecting compositions that reduce amyloid formation.

36. The method of any of Options 20-35, wherein the rate of formation ofamyloid is analyzed or quantified in (c).

37. A kit comprising a microbial amyloid or a microbial amyloidprecursor and α-Synuclein, being present in one or more containerswithin said kit.

38. The kit of Option 37, wherein said microbial amyloid or microbialamyloid precursor comprises CsgA.

39. A method of treating or inhibiting an amyloid disorder in a subjectcomprising:

(a) contacting a plurality of concentrations of a microbial amyloid or amicrobial amyloid precursor with a plurality of concentrations ofα-Synuclein in the presence of a composition;(b) analyzing or measuring the formation of new amyloid after thereaction set forth in (a);(c) comparing the analysis or measurement made in (b) with an analysisor measurement of a control, wherein said control comprises analyzing ormeasuring the formation of amyloid after the reaction set forth in (a)in the absence of said composition; and(d) if the formation of amyloid in the presence of said composition isincreased relative to the formation of amyloid in the absence of saidcomposition, administering to said subject an effective amount of apharmaceutical composition suitable for inhibiting or treating saidamyloid disorder.

40. The method of Option 39, wherein said microbial amyloid or microbialamyloid precursor comprises CsgA.

41. The method of any of Options 39-40, wherein said compositioncomprises tissue, bodily fluid or an extract thereof.

42. The method of any of Options 39-41, wherein said compositioncomprises feces, urine, blood, spinal fluid, or saliva, or a componentthereof.

43. The method of Options 39-42, wherein said pharmaceutical compositioncomprises one or more probiotic bacteria.

44. The method of Options 39-43, wherein said pharmaceutical compositioncomprises one or more bacteria selected from the group consisting ofBacteroides, Prevotella, Parabacteroides, Faecalibacterium, Eubacterium,Roseburia, Blautia, Coprococcus, and Bifidobacterium, or any combinationthereof.

45. The method of Options 39-44, wherein said pharmaceutical compositioncomprises one or more bacteria selected from the group consisting of B.fragilis, B. vulgatus, and B. thetaiotaomicron; or any combinationthereof.

46. The method of any of Options 39-45, wherein the rate of formation ofamyloid is analyzed or quantified in (b).

47. The method of any of Options 39-46, further comprising identifyingor selecting said subject as one that would benefit from a treatment orinhibition of an amyloid disorder.

48. The method of any of Options 39-47, further comprising identifyingor selecting said subject as one at risk of or showing symptoms of oneor more of Parkinson's Disease. Lewy Body Dementia, incidental Lewy bodydisease. Lewy body variant of Alzheimer's disease, multiple systematrophy, pure autonomic failure, or any combination thereof.

EXAMPLES Example 1

To a subject, one or more of the compounds described above (e.g., acomposition comprising a compound of the invention) is administeredorally or rectally on a regular basis, such as daily. Bacterial amyloidformation in the GI tract and/or α-synuclein aggregation levels withinthe GI tissue are monitored by fecal sampling or by biopsy. Therapy iscontinued to prevent bacterial amyloid (curli) formation and/orα-synuclein aggregation. Changes in the patient's GI function and motorsymptoms are monitored. For subjects in which the administration of saidone or more compounds results in reduced formation ofmicrobially-induced amyloid in the gut, improvements in one or more GIsymptoms, one or more motor symptoms and/or one or more neurologicalsymptoms are observed.

Example 2

One or more of the compounds of the invention is obtained or synthesizedand incorporated into an enteric or colon-selective formulations torelease material at site of action and by-pass the stomach and most ofthe small intestine. This provides delivery of the composition at thesite of curli production and/or α-synuclein aggregation, and minimizesabsorption of the composition into systemic circulation.

Example 3

One or more of the compounds of the invention is obtained or synthesizedand incorporated into a formulation for controlled release in the lowersmall intestine or in the colon. This provides for lower and/or lessfrequent dosing, and side effects are minimized. Controlled release inthe lower small intestine or colon may be achieved by any of a varietyof approaches known in the art and includes enteric coated capsules,tablets, soft gels, intrinsically enteric capsules, multi-layeredformulations, coated micronized forms of the polymeric material, and thelike.

Example 4

A subject is administered a combination of more than one of thecompounds of the invention. Combining a curli inhibitor with anα-synuclein aggregation inhibitor blocks aggregation at two criticalpoints simultaneously. For subjects in which the administration of saidone or more compounds results in reduced formation ofmicrobially-induced amyloid in the gut, improvements in one or more GIsymptoms, one or more motor symptoms and/or one or more neurologicalsymptoms are observed or measured.

Example 5

The Thy1-α-synuclein (α-synuclein-overexpressing [ASO]) mouse displaysprogressive deficits in fine and gross motor function, as well as, gutmotility defects. Evidence has linked unregulated α-synuclein expressionin humans to a higher risk of PD, providing an epidemiologicalfoundation for the Thy1-α-synuclein mouse model. Defects in coordinatedmotor tasks become evident at 12 weeks of age. Motor function ismeasured via four tests: beam traversal, pole descent, nasal adhesiveremoval, and hind limb clasping reflexes, as previously validated inthis model (described in Fleming et al., J. Neurosci. 24, 9434-9440(2004), and Sampson et al., Cell 167(6):1469-1480 (2016) the content ofwhich are hereby expressly incorporated by reference in its entirety).ASO mice require significantly more time to cross a challenging beamcompared to wild-type littermates and also exhibit increased time todescend a pole, two measures of gross motor function. Removal of anadhesive from the nasal bridge, a test of fine motor control, is alsoimpaired in SPF-ASO mice compared to SPF-WT mice, as is the hind limbclasping reflex, a measure of striatal dysfunction.

ASO neonates are divided into two groups. To one group is administeredone or more compositions as described above, and the other is untreatedor mock-treated. Compositions are administered daily for 12-13 weeks. At13 weeks and thereafter, motor skills are evaluated. ASO mice treatedwith the compositions described above require less time to cross achallenging beam, decreased time to descend a pole, enhanced removal ofan adhesive from the nasal bridge, and an enhanced hind limb claspingreflex relative to untreated ASO mice.

Fecal pellets are also obtained from test animals. Fecal pellets fromtreated ASO mice show lower levels of bacterial adhesive pili (curli),as well as, lower levels of aggregated α-synuclein relative to untreatedASO animals.

After 16 weeks, animals are sacrificed and their brain enteric nervoustissue is analyzed for the presence of α-synuclein aggregates. Utilizingan antibody that recognizes only conformation-specific α-synucleinaggregates and fibrils, immunofluorescence microscopy is performed tovisualize α-synuclein inclusions. Notable aggregation of α-synuclein isobserved in the caudoputamen (CP), substantia nigra (SN), and entericneurons of untreated ASO animals relative to levels seen in treatedanimals. Western blots of brain extracts are also performed.Significantly less insoluble α-synuclein is found in brains and entericnervous tissue of treated ASO animals.

Example 6

Roles of functional amyloid formation in curli-driven pathophysiologywere examined in mice using techniques described in co-pendingapplication PCT/US20181032605, the entire content of which are hereinincorporated by reference. As an initial matter, effects ofepigallocatechin gallate (EGCG) on biofilm growth by wild-type E. coliwere examined, along with effects of EGCG on αSyn amyloid formation invitro. FIG. 1A is a graph showing Crystal violet staining of biofilmgrowth by wild-type E. coli following 4 days in static culture, withindicated concentrations of EGCG; data assessed by optical density (OD).FIG. 1B is a graph showing in vitro αSyn aggregation measured byThioflavin T fluorescence during αSyn amyloid formation alone or in thepresence of CsgA (25:1 molar ratio), with and without EGCG (50 μM)treatment.

Germ-free Thy1-αSyn mice (ASO) mono-colonized with WT E. coli at 5-6weeks of age, and given water alone (Vehicle: Veh) or treated with EGCGad lib in drinking water (+EGCG). RNA was extracted from fecal pelletsand csgA expression quantified by qRT-PCR, relative to rrsA. FIG. 1C isa graph showing fold-change in csgA expression. Motor function wasassessed at 15-16 weeks of age by quantifying beam traversal time (FIG.1D), pole descent time (FIG. 1E), nasal adhesive removal time (FIG. 1F),hindlimb clasping score (FIG. 1G), and wire hang tests (FIG. 1H). Timeto cross, time to descent, time to remove, and hindlimb score were lowerin the EGCG-treated mice, while time to fall was higher in theEGCG-treated mice compared to vehicle treated control.

FIG. 1I is a graph showing principal component analysis of compiledmotor scores from tests in (FIGS. 1D-H). FIGS. 1J-K are a series ofgraph showing Proteinase K resistant αSyn aggregates (indicated by whitearrows) in the substantia nigra imaged via immunofluorescencemicroscopy. Shown are vehicle-treated (FIG. 1J) and EGCG-treated mice(FIG. 1K). Levels of Proteinase K resistant αSyn aggregates were lowerin the EGCG-treated mice than in untreated controls. Thus, assessment ofmotor performance reveals that EGCG treatment in accordance with someembodiments herein successfully dampens progressive motor deficitsexacerbated by E. coli, while also preventing αSyn aggregation in boththe striatum and midbrain.

FIGS. 1L-M show quantification of insoluble αSyn fibrils in the striatum(FIG. 1L) and ventral midbrain (FIG. 1M) by dot blot assay. Thinsections of brain were stained for Iba1 (microglia), 3D cellularreconstructions generated, and morphological characteristics quantifiedfrom microglia resident in the striatum (FIG. 1N) and substantia nigra(FIG. 1O), n=3 (FIGS. 1A, 1B, 1N, 1O), n=8 (FIG. 1C), n=10-11 (FIGS.1D-I), n=4 (FIGS. 1L-M). Points represent individuals, bars representthe mean and standard error. Data analyzed by one-way ANOVA with Tukeypost-hoc test for FIG. 1A, two-tailed Mann-Whitney for FIGS. C-K, ortwo-tailed t-test for FIG. 1L. For FIGS. 1A-1L *p≤0.05; **p≤0.01;***p≤0.001. Motor data are compiled from 2 independent cohorts.

Accordingly, it is shown that in vivo treatment with compounds inaccordance with compositions and methods in accordance with someembodiments herein inhibit or reduce αSyn amyloid formation in vitro.Furthermore, these compounds improved motor scores, consistent withinhibition, amelioration, and alleviation of symptoms ofaggregate-related diseases such as parkinsonism in accordance with someembodiments herein.

Example 7

Additional experiments, using techniques described in co-pendingapplication PCT/US2018/032605, the entire content of which are hereinincorporated by reference, showed that mono-colonization withcurli-sufficient bacteria induce increased αSyn-dependent pathology andinflammatory responses in the brain. Germ-free (GF) wild-type (WT) orThy1-αSyn (ASO) mice were mono-colonized with either wild-type,curli-sufficient E. coli (WT) or curli-deficient E. coli (ΔcsgBAC). FIG.2A is a graph showing total αSyn in whole brain lysates quantified byELISA. FIG. 2B is a graph showing quantification of insoluble αSynfibrils in the striatum by dot blot assay. FIGS. 2C-D showquantification of TNFα (FIG. 2C) and IL-6 (FIG. 2D) by ELISA from thestriatum. FIGS. 2E-G show the results of staining thin sections ofbrains derived from ASO mice were stained for Iba1 (microglia), 3Dcellular reconstructions generated, and morphological characteristicsquantified of microglia resident in the striatum, n=3 (FIGS. 2A-B),n=6-7 (FIG. 2C, 2D), n=4 (FIGS. 2E-G) (averaged from 20-40 cells fordiameters, or 5-7 cells for branching). Points represent individuals,bars represent the mean and standard error. Data analyzed by one-wayANOVA with Tukey post-hoc test for FIGS. 2A-D, or two-tailed t-test forFIGS. 2E and 2F *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Consistentwith this effect of curli-sufficient bacteria on mouse models, it isshown that the relative abundance of csgA is increased in the gut ofhuman Parkinson's Disease (PD) patients. Relative abundance of csgA wasdetermined by PICRUSt analysis of available 16S RNA data from humanfecal samples (ENA Accessions: PRJNA268515, PRJEB4927, and PRJEB14674).Based on this analysis, it was observed that relative abundance of csgAwas higher in the gut of the PD patients (FIG. 2H). Furthermore,wild-type (FIG. 2I) or Thy1-αSyn (ASO) (FIG. 2J) mice were colonizedwith microbes derived from persons with PD or matched controls (ENAAccession: PRJEB17694), and PICRUSt imputed analysis of 16s rRNAsequences indicated greater abundance in the PD-transplanted microbiomescompared to healthy controls (FIG. 2K). For FIGS. 2H-J, points representindividuals, bars represent the mean, data analyzed by two-tailedMann-Whitney test. *p≤0.05; **p≤0.01. Thus, it is observed that thepresence or elevated levels (compared to healthy controls) of bacterialproteins such as csgA in the gut correlates with amyloid disorders,including PD.

Example 8

Additional experiments show that that intestinal curli promotesprogressive synuclein-dependent pathophysiology. Conventionally-raisedThy1-αSyn (ASO) animals were injected intestinally with 30 μg ofsynthetic CsgA hexamer (CsgA; N-QYGGNN-C) or non-amyloidogenic peptide(N122A; N-QYGGNA-C). Each peptide spanned the aggregation domain ofCsgA. Motor and GI function tested over time at 0, 7, 21, and 70 dayspost-injection in the beam traversal (FIG. 3A), pole descent (FIG. 3B),adhesive removal (FIG. 3C), hindlimb clasping score (FIG. 3D), wire hang(FIG. 3E, fecal output (at day 70) (FIG. 3F). FIG. 3G is a graphdepicting principal component analysis of compiled motor scores of FIGS.3A-F. FIGS. 3H-I depict quantification of insoluble αSyn fibrils in thestriatum (FIG. 3H) and ventral midbrain (FIG. 3I) by dot blot assay, n=8(FIGS. 3A-G), n=4 (FIG. 3H). Points represent individuals, barsrepresent the mean and standard error. Time courses analyzed by two-wayANOVA, with Sidak post-hoc test for between group comparisons indicatedabove individual time points, and brackets indicating significancebetween treatments. Data in FIG. 3H were analyzed by two-tailedMann-Whitney test. For FIGS. 3A-I, *p≤0.05; **p≤0.01; ***p≤0.001;****p≤0.0001. Compilation of motor performance by PCA indicates asymptomatic shift in mice injected with the CsgA peptide compared tocontrols, demonstrating that the overall motor function of these animalshas been impaired (FIG. 3G). Furthermore, increased αSyn fibrils aredetected in the midbrains of amyloidogenic CsgA-injected animals (FIG.3H), demonstrating alterations to central nervous system (CNS) pathologyfollowing amyloid administration directly to the GI tract. Thus, gutexposure to a CsgA peptide capable of forming amyloids is sufficient toexacerbate long-lasting motor deficits in αSyn over-expressing mice.

Thus, it was shown herein that intestinal curli increased time to cross,time to descend, time to remove, and hindlimb score, and decreased timeto fall and fecal pellets per mouse. The increases in time to cross,time to descend, and hindlimb score, and decrease in fecal pellets permouse were statistically significant at the noted levels (See FIGS. 3B-Dand 3F). Accordingly, it is contemplated that intestinal curli caninduce symptoms of amyloid disorders in vivo.

Example 9

To further explore cause-and-effect relationships between the microbiomeand PD, it was studied whether production of curli by an otherwisehealthy human microbiome is sufficient to impair motor performance. GFASO mice were transplanted with microbiota from a healthy human donorpredicted to contain low levels of CsgA-producing bacteria, as indicatedby PICRUSt analysis following 16S rRNA sequencing. Fecal abundance ofamyloid-producing bacteria in these humanized animals was studied. Humansamples from previous cohort (ENA Accession: PRJEB17694) were analyzedby PICRUSt to infer abundance of csgA encoded within each population(FIG. 4A). Fecal pellets of Thy1-αSyn (ASO) mice receiving healthy-humanderived fecal microbes enriched with cither wild-type, curli-sufficientE. coli (WT) or curli-deficient E. coli (ΔcsgBAC) were analyzed by b,qPCR for rrsA abundance relative to 16s rRNA present in fecal bacterialDNA and by c, qPCR analysis for csgA expression relative to rrsA infecal bacterial RNA. n=5 (FIG. 4A), n=8 (FIGS. 4B-C). Points representindividuals, bars represent the mean and standard error. Data wereanalyzed by two-tailed Mann-Whitney test. ***p≤0.001.

Thus, it is shown that intestinal levels of amyloid-producing microbialorganisms can be detected in samples in accordance with some embodimentsherein, and moreover, intestinal levels of amyloid-producing microbialorganisms have been shown differ in subjects having amyloid aggregatesthat model an amyloid disorder (compared to healthy controls).

Example 10

Inhibition of functional amyloid formation was studied. Germ-freeThy1-αSyn mice (ASO) were monocolonized with wild-type E. coli andtreated with water alone (Vehicle, Veh) or given EGCG ad lib in drinkingwater (+EGCG). Motor function was assessed at 10, 12, and 15 weeks ofage by quantifying beam traversal time (FIG. 5A), pole descent time(FIG. 5B), nasal adhesive removal time (FIG. 5C), hindlimb claspingscore (FIG. 5D), and wire hang tests (FIG. 5E). FIG. 5F depictsprincipal component analysis of compiled motor scores from FIGS. 5A-D.Thin sections of brain were stained for Iba1 (microglia) andmorphological characteristics quantified of microglia resident in thestriatum (FIGS. 5G-H) and substantia nigra (FIGS. 51 -J). N=10-11 (FIGS.5A-F), n=3 (FIGS. 5G-J) (averaged from 5-7 cells for branching). Barsrepresent the mean and standard error. Time courses analyzed by two-wayANOVA, with Sidak post-hoc test for between group comparisons indicatedabove individual time points, and brackets indicating significancebetween treatments. Data in (FIGS. 5G-J) analyzed by two-tailed t-test.*p≤0.05; **p≤0.01; ****p≤0.0001.

Accordingly, inhibition of functional amyloid formation in accordancewith some embodiments herein dampens progressive motor deficits. Withoutbeing limited by theory, it is contemplated that curli produced by E.coli utilize an amyloid-dependent pathway to exacerbate hallmark motordeficits and pathologies of PD in this preclinical model. It is furthercontemplated that inhibition of bacterial amyloid production, formationand/or interaction with mammalian amyloids in accordance with someembodiments herein is a useful intervention of neurodegenerativeconditions caused by protein aggregation, for example amyloid disordersas described herein.

Example 11

Effects of the bacterial amyloid protein. CsgA on the seeding of αSynfibrillization were studied. In vitro biophysical analysis was conductedwith purified αSyn and CsgA proteins. It was tested whether the majorcurli subunit, CsgA, is capable of cross-seeding the formation of αSynaggregations. It was observed that addition of purified CsgA tomonomeric αSyn in vitro results in significantly accelerated productionof αSyn aggregates (FIGS. 6A-B). FIG. 6A shows aggregation as measuredby Thioflavin T fluorescence over time during αSyn amyloid formationalone or in the presence of CsgA monomers (25:1 molar ratio, yellow).FIG. 6B shows time to reach exponential fibrillization, lag phase. FIGS.6C-H are a series of representative transmission electron micrographs ofαSyn alone (FIGS. 6C, 6F) or CsgA alone (FIGS. 6E, 6H), or incombination (FIGS. 6D, 6G), at 0 hours (FIGS. 6C-E) and 60 hours (FIGS.6F-H) post-aggregation. FIGS. 6I-K are a series of graphs illustratingcircular dichroism spectroscopic analysis of αSyn fibrillization aloneor in the presence of CsgA at 0, 12.5, and 60 hours post-aggregation.For FIG. 6A and FIG. 6B, n=3. Bars represent the mean and standarderror. Data are analyzed by two-tailed, t-test. **p≤0.01. Data arerepresentative of 2 independent trials. Thus, the bacterial amyloidprotein. CsgA, in accordance with some embodiments herein seeds αSynfibrillization. Without being limited by theory, it is contemplated thatCsgA not only accelerates the generation of αSyn aggregates in vitro,but these subsequent αSyn structures maintain pathogenic attributes,similar to observations with other amyloids that propagate in aprion-like manner.

Example 12

Effects of CsgA on seeding synuclein aggregation and propagation wasstudied through transient interactions.

FIG. 7A is a graph showing thioflavin T fluorescence during αSyn amyloidformation alone or in the presence of 5% seeds previously generated byaddition of CsgA monomer to αSyn (as in FIG. 2A) or αSyn alone. FIGS.7B-F are a series of transmission electron micrograph of fibrilstructures generated by the addition of above seeds and of seedsthemselves. FIG. 7G is a graph showing surface plasmon resonancemeasurements of surface immobilized αSyn with additions of either CsgAmonomer or seeds, or DOPS-DOPG cholesterol as positive control. Thus, itis shown that the final CsgA-induced synuclein fibrils purified fromcompleted biochemical reactions maintain an ability to accelerate αSynamyloid formation. Without being limited by theory, these results areconsistent with transient interactions or interactions betweenoligomeric forms of the proteins at later stages in amyloidogenesis(FIG. 7G).

Accordingly, CsgA was shown to seed synuclein propagation throughtransient interactions.

Example 13

A library of potential amyloidogenesis inhibitors is obtained. Suchlibraries may be found in preexisting repositories, or may be generatedde novo by, for example, combinatorial synthesis or by solid phasepeptide synthesis utilizing such methods as are well known in the art.See, for example. Jensen, K. J. et al., eds. Peptide Synthesis andApplications, 2^(nd) Edition, 2913, which is incorporated by referenceherein for its teachings of solid phase peptide synthesis, combinatorialpeptide synthesis, and the generation of peptide libraries. Naturalproduct libraries may also be utilized. In a multi-well assay plate, abacterial amyloid initiator, such as E. coli CsgA is placed in varyingconcentrations in one dimension, while a host-derived amyloidogenicprotein, such as α-synuclein, is placed in varying amounts in the seconddimension, such that each well contains a different ratio of amyloidinitiator and amyloid precursor. To each well, a constant amount of anindicator of amyloid formation, such as thioflavin (ThT), is added, aswell as a constant amount of an individual test compound. Each tray isagitated to initiate amyloid formation, and thioflavin fluorescence ismonitored. Compounds that show deviations in the rate of fluorescencedevelopment over time will be identified as candidates that enhance orinhibit amyloid formation.

Example 14

A suspected amyloidogenesis inhibitor is combined with a bacterialamyloid initiator and an amyloid precursor in the presence of ThioflavinT (ThT). Separately, as a control, bacterial amyloid initiator, amyloidprecursor, and Thioflavin T are combined in the absence of the suspectedamyloidogenesis inhibitor. Thioflavin T fluorescence is monitored overtime. A reduction in the rate of increase in Thioflavin T fluorescence,and/or a reduction in the maximum level of Thioflavin T fluorescence inthe sample containing the suspected inhibitor, relative to the controlsample, confirms that the suspected amyloidogenesis inhibitor is in factfunctioning to inhibit amyloid formation.

Example 15

A sample of tissue, fluid, feces, or intestinal contents is collectedfrom a subject. Said sample is combined with a bacterial amyloidinitiator, such as E. coli CsgA, a host-derived amyloidogenic protein,such as α-synuclein and an indicator of amyloid formation, such asThioflavin T (ThT). Thioflavin T fluorescence is monitored. An increasein fluorescence consistent with an increase in the rate of formation ofamyloid in the presence of said sample, relative to the rate of amyloidformation in the absence of said sample, indicates an increase in therisk of α-synucleinopathy, including Parkinson's disease and/or Lewybody dementia. This increased risk is further correlated with resultsfrom conventional neurological examinations in order to calculate adefined risk of commencement and/or progression of an α-synucleinopathyor other neurodegenerative disorder implicating amyloid formation.

Example 16

In non-binding, black plastic, 96-well plates, 50-100 μM α-synuclein isincubated in 0.01M Tris buffer (pH 7.4) or 0.05 M potassium phosphatebuffer pH 7.3 in the presence of 12 μM of Thioflavin T (prepared inwater). Purified CsgA monomer in 0.05 M potassium phosphate buffer, pH7.3 is added to each well at a molar ratio of 1:10, 1:25, 1:50, or1:100. Inhibitory compounds are prepared in appropriate bufferedsolutions based on solubility, such as 0.05 M potassium phosphatebuffer, pH 7.3 or DMSO. Compounds and appropriate buffer controls areadded to α-synuclein- and CsgA-containing wells, to a final volume of150 μL per well. The concentration of each compound is dependent on thetype of compound being screened but generally is expected to fall withinthe range of 1 μM to 200 μM in initial screens. Details regarding theaddition of such compounds depend on the types of compounds available inthe accessible small molecule libraries. Independent wells containingα-synuclein alone and CsgA alone serve as specificity controls, or incombination in the absence of potential inhibitors. A single, sterilizedglass or Teflon bead with a ˜1-2 mm diameter is added to each well. Theplate is incubated within a fluorescent-capable microplate reader withcontinuous orbital shaking (˜100-250 rpm) at 37° C. Fluorescence ismeasured every 1-2 hours with an excitation of 440±10 nm and emission of490±10 nm. Measurements are taken over a 24-72 hour period. Asα-synuclein amyloids form, emission spectra hit maximum intensity ˜24-48hours under these conditions following a sigmoidal curve. After thistime, emission intensity can decrease as amyloids become insoluble andnon-fluorescent.

Amyloid formation appears over 3 phases (See, e.g., FIG. 1B): (1) a lagphase whereby fluorescence intensity is low occurring over the first˜0-24 hrs; (2) A log phase whereby fluorescence intensity increaseslogarithmically from ˜2-48 hrs; and (3) a plateau phase wherebyfluorescence intensity hits a maximum and either remains unchanged forthe remaining time period or begins to decrease due to insolubleα-synuclein precipitating out of solution. Maximum intensity occursbetween 24-72 hours.

Aggregation kinetics, as measured by thioflavin fluorescence, in thepresence of compounds can be normalized to the kinetics observed withα-synuclein and CsgA alone. Potential inhibitors may act to lengthen thelag phase, decrease the rate of change during the log phase, decreasethe maximum intensity reached, or any combination thereof.

Once initial candidates are identified, dose responses over a wide-rangeof concentrations can be determined, as well as specificity againstCsgA:synuclein aggregates, or CsgA and α-synuclein individually. In somevariations of this screen. CsgA:synuclein aggregates can be monitoreduntil the log phase, and potential inhibitory compounds introduced atthis time. Subsequently, inhibitors which can act once amyloid formationis already in process can be identified (See, e.g., FIG. 1B).

Example 17

The assay is practiced as described in Example 16, in which fullconcentration curves are generated for each compound. This enablesaccurate determination of the EC50 for each compound and can exposecertain compounds limitations (e.g., expose compounds that do not leadto complete inhibition of aggregation).

Example 18

The assay is practiced as described in Example 16, and is formatted forhigher throughput screening in a variety of ways. For example, ratherthan a full concentration curve for each compound, a three-pointconcentration curve is used to distinguish compounds with adose-response effect from those with a non-specific andconcentration-independent effect. For even higher throughput screening,the assay is formatted in 96-well, 384-well or 1536-well plates andcompounds are tested at a single concentration (e.g., 1 μM) and at asingle timepoint (e.g., 24-72 hours). This enables the observer todistinguish potential candidates from compounds with no effect or withno effect at a relevant concentration.

Example 19

The assay is practiced as described in Example 16, in which full timecourse curves are generated for each compound. Time-course curves showwhether a compound inhibits in a linear or sigmoidal fashion over time,and/or whether the complete inhibition can be achieved with a givencompound.

Example 20

The assay is practiced as described in Example 16, and is furthermodified to assess mechanistic processes and compound activity in a moredynamic environment in which both α-synuclein (or other host amyloidprotein) and curli (or other bacterial amyloid) are present in theassay. The observer then assesses the ability of compounds to inhibitaggregation of one or the other proteins in the presence of anaggregation template. For example, bacterial amyloid component CsgA isknown to promote and/or accelerate α-synuclein aggregation. In an invivo environment, a candidate compound with α-synuclein aggregationinhibition activity is exposed to an aggregation promoting or templatingactivity from bacterial amyloid. Thus, formatting the assay by inclusionof both monomeric α-synuclein (or other host amyloid) and aggregatedbacterial amyloid allows assessment of drug candidates in a morephysiologically relevant in vitro environment.

Example 21

The assay is practiced as described in Example 16, wherein the methodfurther comprises combining a curli (bacterial amyloid) aggregationinhibitor with an α-synuclein aggregation inhibitor. This combinationhas the added benefit of blocking aggregation at two critical pointssimultaneously. The assay utilizes the monomeric forms of bothα-synuclein and curli (CsgA), and measurements analogous to those shownin FIG. 1B are obtained.

Example 22

The assay is practiced as described in Example 16, wherein the methodfurther comprises combining a curli (bacterial amyloid) dis-aggregationpromoter with a promoter of α-synuclein dis-aggregation. Thiscombination has the added advantage of effecting dis-aggregation at twocritical points simultaneously. The assay utilizes the fully aggregatedforms of both α-synuclein and curli, and measurements analogous to thoseshown in FIG. 1B are obtained.

Example 23

The assay is practiced as described in Example 16, wherein the methodfurther comprises combining a curli (CsgA, bacterial amyloid)aggregation inhibitor with an α-synuclein dis-aggregation promoter. Thiscombination has the added advantage of inhibiting the nucleation ororigination of amyloid while simultaneously effecting dis-aggregation ofan already initiated process. The assay utilizes the fully aggregatedform of α-synuclein and the monomeric form of curli (CsgA), andmeasurements analogous to those shown in FIGS. 2A-3I are obtained.

Example 24

The assay is practiced as described in Example 16, wherein the methodfurther comprises combining a curli (bacterial amyloid) dis-aggregationpromoter with an α-synuclein aggregation inhibitor. This combination hasthe added advantage of destroying pathogenic bacterial amyloid whilesimultaneously inhibiting α-synuclein aggregation. The assay utilizesthe monomeric form of α-synuclein and the fully aggregated form ofcurli, and measurements analogous to those shown in FIGS. 2A-3I areobtained.

Example 25

The assay is practiced as in any of Examples 16-24, except that a CsgAmutant that is incapable of aggregation is included. Compounds that relyon the presence of structured (aggregated) CsgA in these processes willshow reduced effectiveness in this version of the assay.

Example 26

Human α-synuclein was expressed in E. coli BL21(DE3) from a plasmidderived from pT7 or pET11a into which the full-length gene for untaggedhuman α-synuclein was inserted. Cells were induced at OD600 0.6 with 0.8mM WTG for 4 hours, harvested by centrifugation and suspended in avolume of lysis buffer (10 mM Tris, pH 8.0, 1 mM EDTA, and 1 mM PMSF)equivalent to one-tenth the volume of culture, and lysed by boiling for20 minutes. Cell debris was pelleted by centrifugation, clarified lysatewas treated with 10%, streptomycin sulfate at 136 μL/mL, followed byglacial acetic acid at 228 μL/mL, lysate was centrifuged, supernatantwas transferred to a fresh tube and protein was precipitated viaaddition of an equal volume of saturated (100%) ammonium sulfate andincubated at 4° C. on a rocking shaker for 1 hour. The protein waspelleted via centrifugation and washed with an equal volume of 100 mMammonium acetate in chilled ethanol, pelleted via centrifugation, washedtwice with chilled ethanol, dried overnight, resuspended in 50 mMpotassium phosphate buffer pH 7.3 or 10 mM Tris pH 7.4, and passedthrough a 50 kDa cut-off column. Immediately prior to use, the purifiedα-synuclein was filtered with a 0.2 μm nylon filter. The α-synucleinconcentration was determined using the absorption at 280 nm or BCAassay.

Example 27

Full length, recombinant CsgA monomers can be prepared as described fromZhou et al. (2012). Journal of Biological Chemistry 287(42). Briefly.CsgA is cloned into a pET11d vector containing a C-terminal 6× His tag.Following growth in rich media, CsgA production is induced at an OD600of approximately 0.9 by 0.5 mM IPTG at 37° C. for 1 hr. Bacteria werelysed in 8M guanidine hydrochloride in 50 mM potassium phosphate buffer,pH 7.3 overnight or for approximately 1-2 hours at room temperature on arocking platform. After centrifugation at 10,000×g for 20 minutes, thesupernatant was sonicated on ice for 6 10 second intervals, incubatedwith nickel-nitrilotriacetic acid resin (Sigma) at room temperature for1 h and then loaded onto a disposable polypropylene column (Thermo). Thecolumn was washed with 50 mM potassium phosphate buffer, pH 7.3 and 50mM potassium phosphate buffer pH 7.3 containing 12.5 mM imidazole.Proteins were eluted with 50 mM potassium phosphate buffer, pH 7.3containing 125 mM imidazole. To get monomeric CsgA, fractions containingthe target protein were combined and loaded onto a 30-kDa centrifugalfilter unit (Thermo) to remove dimers and other oligomers. Purified CsgAwas passed through a pre-chilled desalting column (Zeba) to removeimidazole.

Alternatively, synthetic hexapeptides of CsgA consisting of thesequence: Nterm-QYGGNN-Cterm, are commercially available fromBio-synthesis. Inc.

Example 28

Another alternative is to utilize pre-formed CsgA amyloid seeds, bypreparing purified curli extracts from biofilms as described inCollinson et al. (1991). Journal of Bacteriology. 173(15). Wild-typeEscherichia coli is grown on YESCA media with or without Congo Red dyeadded, for 3-7 days at room temperature. The cultures are scraped into10-30 mLs of 10 mM Tris, pH 8. Cells are lysed by sonication or byfreeze-thaw. Cell lysates are treated with 0.1 mg RNase A, 0.1 mg DNaseI and MgCl₂ added to 1 mM, and incubated for 20-30 min at 37 C. Lysozymeis added to 1 mg/mL and further incubated at 37° C. for 20-40 min. SDSis added to 1% and incubated at 37° C. for 20-40 min. Insoluble materialis collected by centrifugation at 12.000×g for 15 min. Samples arere-suspended in 1-10 mL Tris buffer, boiled at 90° C. for 15 min, andthe above processes are repeated (Digestion with RNase, DNase, Lysozyme,and SDS treatment). Samples are washed twice with Tris buffer,resuspended in Laemli buffer, boiled, and loaded onto an SDS-PAGE gel(4-20%). Samples are electrophoresed at 20 mA for 5 hrs. The remaininginsoluble material in the stacking gel is collected, washed three timeswith water, washed twice with 95% ethanol, and dried. The sample isresuspended in 0.2M glycine pH 1.5 and boiled for 10-15 min. Insolublematerial is collected by centrifuging at 16 k×g for 10 min. Theinsoluble material is washed five times with water, and resuspended inPBS. Finally, the sample is sonicated by electrode or water bath for 1hour before protein content determined by BCA or absorbance at 280 nm.

Such alterations may change the kinetics of aggregation, theconcentrations of compounds needed to inhibit aggregation, the ratiosrequired to display CsgA-mediated synuclein aggregation, or combinationsof the above.

Example 29

In other iterations of the protein purifications used in the assays ofExamples 27 and 28, CsgA may be produced without a histidine tag or withan alternate tag, and it may contain a sequence to promote its excretionfrom the cell. α-synuclein may be engineered to contain a histidine tagor other tag to promote purification by affinity for example toimmobilized metals such as nickel. CsgA and α-synuclein may be purifiedusing alternate methods familiar to one skilled in the art, such asammonium sulfate precipitation with alternate concentrations of ammoniumsulfate in a single step or in multiple steps with increasingconcentrations of ammonium sulfate; alternately, ammonium sulfateprecipitation may be omitted. Alternate resins or materials to separateCsgA or α-synuclein from other proteins based on protein affinity,cation exchange, anion exchange, hydrophobic interactions, multiplemodes or mixed modes may be used, as are familiar to one skilled in theart. Protein separation may be performed using batch purification,pre-packed columns, gravity flow, low pressure, high pressure, and highpressure liquid chromatography, using methods familiar to one skilled inthe art, and the methods may be used individually or in combination.CsgA or α-synuclein may be separated from other proteins on the basis ofsize using methods familiar to one skilled in the art, such as sizeexclusion chromatography or high pressure liquid chromatography. CsgAand α-synuclein may be purified under denaturing conditions for all orpart of the assay purification process, with alternate concentrations ofguanidinium hydrochloride or with alternate denaturants at variousconcentrations known to one skilled, such as urea. Alternately, CsgA andα-synuclein may be purified under native conditions familiar to oneskilled in the art; under native conditions or with steps usingnon-denaturing buffers, CsgA is generally purified rapidly to avoidaggregation during the purification process. His-tagged CsgA may beeluted from immobilized metal affinity materials such as Ni-NTA usingalternate methods known to one skilled in the art, such as decreasing pHor addition of chelators such as ethylenediaminetetraacetic acid. Inaddition to or instead of immobilized metal affinity chromatographybased on the affinity of histidine residues in the protein toimmobilized nickel, alternate immobilized metal affinity chromatographyor batch purification methods may be used, such as materials withimmobilized copper, zinc, cobalt or nickel interacting with histidine oralternate amino acids in the protein, such as cysteine or tryptophan, asknown to one skilled in the art. As known to one skilled in the art,alternate buffers may be used with Ni-NTA agarose, such astris(hydroxymethyl)aminomethane, (“Tris”);4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, (“HEPES”);3-(N-morpholino)propanesulfonic acid, (“MOPS”), optionally includingsodium chloride, potassium chloride or other salts, and variousdetergents and reducing agents of compositions and concentrationscompatible with Ni-NTA agarose chromatography or batch purification.

In the assays, alternate concentrations of dimethyl sulfoxide may beused, and concentrations of dimethyl sulfoxide significantly elevatedabove or decreased below 1% may affect the aggregation kinetics of CsgAand α-synuclein. Alternate concentrations of Thioflavin T may be used inthe assay and may affect the fluorescent signal and sensitivity of theassay. Alternate concentrations of CsgA and α-synuclein may be used andsuch alterations may affect aggregation kinetics of α-synuclein and CsgAin the assay. Alternate concentrations of compounds may be tested in theassay, and dose-responses may be evaluated. Additional reagents may beadded to the assay which may affect aggregation kinetics of α-synucleinand CsgA depending on their concentration, including detergents such assodium dodecyl sulfate. Shaking may be included at alternate intervalsin the assay and may affect CsgA and α-synuclein aggregation kinetics. 2mm glass beads may be omitted from the assays including α-synuclein ormay be included in the assays including CsgA, or other sizes orcompositions of beads may be used, and these alterations may affect CsgAand α-synuclein aggregation kinetics. Alternate buffers, such as Tris,HEPES and MOPS, and alternate buffer concentrations may be used in theassay and may affect CsgA and α-synuclein aggregation kinetics. Anyplate reader capable of fluorescent reads with excitation at 438 nM andemission at 495 nm with sufficiently narrow bandwidths, such as 10 nm,may be used. Alternate microplates may be used in the assay, such asblack microplates with clear bottoms. Plates may be sealed withalternate coverings that do not absorb ThT fluorescence, or thecoverings may be removed prior to reads. The fluorescence may be read ata single endpoint or at multiple points over various time intervals, andthe time intervals at which the fluorescence is measured may be constantor may vary during the course of the assay. Other metrics may be used todetermine the effects of the compounds on α-synuclein and CsgAexpression, including examination of Thioflavin T signal over the courseof a kinetic read to determine lag phase in Thioflavin T fluorescence,the shape of a curve produced by the fluorescent signal, and the slopeof the curve.

Example 30

To assess the effects of compounds on aggregation of α-synuclein, acell-free assay was performed using purified α-synuclein and ThioflavinT. In the assay, human α-synuclein was expressed in E. coli BL21(DE3)from a plasmid derived from pT7 or pET11a into which the full-lengthgene for untagged human α-synuclein was inserted. Cells were induced atOD600 0.6 with 0.8 mM IPTG for 4 hours, harvested by centrifugation andsuspended in a volume of lysis buffer (10 mM Tris, pH 8.0, 1 mM EDTA,and 1 mM PMSF) equivalent to one-tenth the volume of culture and lysedby boiling for 20 minutes. Cell debris was pelleted by centrifugation,clarified lysate was treated with 10% streptomycin sulfate at 136 μL/mLfollowed by glacial acetic acid at 228 μL/mL, lysate was centrifuged,supernatant was transferred to a fresh tube and protein was precipitatedvia addition of an equal volume of saturated (100%) ammonium sulfate andincubated at 4° C. on a rocking shaker for 1 hour. The protein waspelleted via centrifugation and washed with an equal volume of 100 mMammonium acetate in chilled ethanol, pelleted via centrifugation, washedtwice with chilled ethanol, dried overnight, resuspended in 50 mMpotassium phosphate buffer pH 7.3 or 10 mM Tris pH 7.4, and passedthrough a 50 kDa cut-off column. Immediately prior to use, the purifiedα-synuclein was filtered with a 0.2 μm nylon filter. The assay wasconducted in 96-well black microplates with a single 1-2 mm glass beadin each well, 20-40 μM Thioflavin T, 1% DMSO. α-synuclein at 50 μM andcompounds at 20-50 μM. Plates were sealed with sealing tape(ThermoFisher 232701), incubated at 37° C. in a Tecan Nano F200 platereader with excitation at 438 nm using a 439 nm filter with a bandwidthof 8 nm, and emission at 495 nm with a 490 nm filter with a bandwidth of10 nm. During the assay, plates were shaken continuously or were shakenfor 999 seconds every 18 minutes. Readings were performed for up to 72hours. Readings were performed hourly for up to 73 hours. The effect ofcompounds on α-synuclein aggregation in the assay was determined withdata from 36 hours. Fluorescence values for each compound withα-synuclein were first adjusted by subtracting the average fluorescenceof the compound in the absence of α-synuclein. These adjusted valueswere normalized by dividing them by the average fluorescence in theno-compound (i.e., untreated) control wells containing α-synuclein, andthe average normalized fluorescence with the compound was expressed as apercentage of the average fluorescence observed in untreated controls.Percent inhibition of α-synuclein aggregation by a compound wasdetermined by subtracting the percent average fluorescence with thecompound at 24 hours from 100%. Results are shown in Table 4. A higherpositive percent inhibition reflects greater inhibition of α-synucleinaggregation, while a negative percent inhibition reflects potentiationof α-synuclein aggregation.

Example 32

In vitro Thioflavin T assay to determine effects of compounds on CsgAaggregation. To assess the effects of compounds on aggregation of E.coli CsgA, a cell-free assay was performed using purified CsgA andThioflavin T. In the assay, histidine-tagged CsgA was over-expressed inE. coli NEB 3016 slyD::kan cells harboring a pET11d vector containingcsgA with the sequence for 6 histidine residues added to the C-terminusand without the See signal (amino acid 1-22) sequence. To induceover-expression of CsgA, 0.5 mM isopropyl β-D-1-thiogalactopyranoside(“IPTG”) was added to cultures with an optical density at 600 nm (OD₆₀₀)of 0.8-1, and induced cells were cultured at 37° C. for 1 hour prior toharvest via centrifugation. Cells were lysed under denaturing conditionswith 8 M guanidine hydrochloride in 50 mM potassium phosphate buffer pH7.3, for 1-2 hours on a rocking platform at room temperature or at 4° C.overnight, and CsgA was purified via immobilized-metal affinitychromatography by batch purification with Nickel-NTA agarose and acombination of low pressure and gravity flow through a disposablepolypropylene column, including washes under low pressure appliedmanually via application of a syringe plunger to the column with 50 mMpotassium phosphate buffer pH 7.3 followed by 12.5 mM imidazole in 50 mMpotassium phosphate buffer pH 7.3, and elution by gravity flow with 125mM imidazole in 50 mM potassium phosphate buffer pH 7.3. Buffers withimidazole were freshly prepared prior to the protein purification.Purified CsgA was passed through a 30 kDa molecular weight cut-offfilter and passed through a desalting column. All steps of the CsgApurification in the absence of guanidine hydrochloride were conducted inrapid succession with as little delay as possible. CsgA was quantifiedvia BCA assay. The assay was conducted in 96-well black microplates with20-40 μM Thioflavin T, 50 mM potassium phosphate buffer pH 7.3, 1% DMSO,CsgA at 6-20 μM, and compounds at 20 μM. Plates were incubated at 25° C.in a Tecan Nano F200 plate reader with excitation at 438 nm using a 439nm filter with a bandwidth of 8 nm, and emission at 495 nm with a 490 nmfilter with a bandwidth of 10 nm, or in a SpectraMax M5 or SpectraMax®i3X with excitation at 438 nM and emission at 495 nm. During the assay,plates were shaken initially for 5 seconds and subsequently for 3seconds prior to fluorescent readings. Readings were performed every 20minutes for up to 24 hours. The effect of compounds on CsgA aggregationin the assay was determined with data from 6-9 hours. Fluorescencevalues for each compound with CsgA were first adjusted by subtractingthe average fluorescence of the compound in the absence of CsgA. Theseadjusted values were normalized by dividing them by the averagefluorescence in the no-compound (i.e., untreated) control wellscontaining CsgA. The median normalized fluorescence from 6-6.5 hours or8-9 hours with the compound was expressed as a percentage of the medianfluorescence observed in untreated controls over the same time period.Percent inhibition of CsgA aggregation by a compound was determined bysubtracting the percent average fluorescence with the compound rom 100%.Results are shown in Table 4. A higher positive percent inhibitionreflects greater inhibition of CsgA aggregation, while a negativepercent inhibition reflects potentiation of CsgA aggregation.

In vitro Thioflavin T assay to determine the effects of compounds onCsgA-seeded G-synuclein aggregation. To assess the effects of compoundson aggregation of α-synuclein seeded by E. coli CsgA, a cell-free assaywas performed using purified α-synuclein, purified CsgA and ThioflavinT. In the assay, histidine-tagged CsgA was over-expressed in E. coli NEB3016 slyD::kan cells harboring a pET11d vector containing csgA with thesequence for 6 histidine residues added to the C-terminus and withoutthe Sec signal (amino acid 1-22) sequence. To induce over-expression ofCsgA, 0.5 mM IPTG was added to cultures with an optical density at 600nm (OD₆₀₀) of 0.8-1, and induced cells were cultured at 37° C. for 1hour prior to harvest via centrifugation. Cells were lysed underdenaturing conditions with 8 M guanidine hydrochloride in 50 mMpotassium phosphate buffer pH 7.3 for 1-2 hours on a rocking platform atroom temperature or at 4° C. overnight, and CsgA was purified viaimmobilized-metal affinity chromatography by batch purification withNickel-NTA agarose and a combination of low pressure and gravity flowthrough a disposable polypropylene column, including washes under lowpressure applied manually via application of a syringe plunger to thecolumn with 50 mM potassium phosphate buffer pH 7.3 followed by 12.5 mMimidazole in 50 mM potassium phosphate buffer pH 7.3, and elution with125 mM imidazole in 50 mM potassium phosphate buffer pH 7.3. Bufferswith imidazole were freshly prepared prior to the protein purification.Purified CsgA was passed through a 30 kDa molecular weight cut-offfilter and through a desalting column. All steps of the CsgApurification in the absence of guanidine hydrochloride were conducted inrapid succession with as little delay as possible. CsgA was quantifiedusing a BCA assay or absorbance at 280 nm with a nanodropspectrophotometer. Human α-synuclein was expressed in E. coli BL21(DE3)from a plasmid derived from pT7 or pET11a into which the full-lengthgene for untagged human α-synuclein was inserted. Cells were induced atOD600 0.6 with 0.8 mM IPTG for 4 hours. Cells were lysed by boiling for20 minutes, cell debris was pelleted by centrifugation, clarified lysatewas treated with 10% streptomycin sulfate at 136 μL/mL and glacialacetic acid at 228 μL/mL, lysate was centrifuged, supernatant wastransferred to a fresh tube and protein was precipitated via addition ofan equal volume of saturated (100%) ammonium sulfate. The ammoniumsulfate pellet was washed with an equal volume of 100 mM ammoniumacetate in ethanol, pelleted via centrifugation, washed twice withethanol, dried overnight, resuspended in 10 mM Tris pH 7.4, and passedthrough a 50 kDa cut-off column. Immediately prior to use, the purifiedα-synuclein was filtered with a 0.2 μm nylon filter. The assay wasconducted in 96-well black microplates with a single glass 1-2 mm beadper well, 20-40 μM Thioflavin T, 1% DMSO, 2 μM CsgA, 50-60 μMα-synuclein, 100 mM sodium chloride, 9.3 mM potassium phosphate pH 7.3,and compounds at 50 μM. Compounds were tested in triplicate. Plates weresealed with sealing tape (ThermoFisher 232701), incubated withcontinuous shaking at 37° C. in a Tecan Nano F200 plate reader, withexcitation at 438 nm using a 439 nm filter with a bandwidth of 8 nm, andemission at 495 nm with a 490 nm filter with a bandwidth of 10 nm, or ina SpectraMax M5 or SpectraMax® i3X with excitation at 438 nM andemission at 495 nm, and readings were performed hourly for up to 73hours. The effect of compounds on CsgA-seeded α-synuclein aggregation inthe assay was determined with data from 17, 18 or 50-52 hours.Fluorescence values for each compound with CsgA and α-synuclein werefirst adjusted by subtracting the average fluorescence of the compoundin the absence of CsgA and α-synuclein. These adjusted values werenormalized by dividing them by the average fluorescence in theno-compound (i.e., untreated) control wells containing CsgA andα-synuclein, and the average or median normalized fluorescence with thecompound at 17, 18 or 50-52 hours was expressed as a percentage of theaverage or median fluorescence observed in untreated controls at thesame timepoints. Percent inhibition of CsgA-seeded α-synucleinaggregation by a compound was determined by subtracting the percentaverage or median fluorescence with the compound at from 100%. Resultsare shown in Table 4. A higher positive percent inhibition reflectsgreater inhibition of CsgA-seeded α-synuclein aggregation, while anegative percent inhibition reflects potentiation of CsgA-seededα-synuclein aggregation.

TABLE 4 Inhibition of Aggregation Compound αSyn CsgA-seeded αSyn CsgA 1 8% (−) 2   93% (+++)  63% (+++)  45% (++) 3 −11% (−−) 4  43% (++) 5 22%(+) −4% (−) 6  36% (++) >90% (+++)  40% (++) 7  2% (−) 8 18% (+) 13% (+)9 −33% (−−) 11 −24% (−−) 12 −23% (−−) 13 −16% (−−) 14  3% (−)  74% (+++)15  52% (++) 16  31% (++) 17 18% (+) 18 16% (+) 19 29% (+) 14% (+)  18%(+) 20 11% (+)    61% (+++) 21 −1% (−)  4% (−) 22  58% (++) 23 −22%(−−)  −4% (−) 24 −27% (−−)  −1% (−) 25 17% (+) −150% (−−)  26 24% (+)<−50% (−−)    8% (−) 27  49% (++)  79% (+++)  44% (++) n = 1 28 20% (+)<−50% (−−)   −6% (−) 29  9% (−) 30 −56% (−−) 31 −20% (−−) 32  48%(++) >90% (+++)  53% (++) 33   92% (+++)  59% (++) 34 −11% (−−) 35 −53%(−−) 36 −47% (−−) 37 −17% (−−) 38 14% (+) 39 −4% (−) 40 −12% (−−) 15%(+) 41 17% (+) 18% (+) 42   98% (+++) >90% (+++)   114% (+++) 43  63%(++)   90% (+++) 44   98% (+++) >90% (+++)   64% (+++) 45 −18% (−−) 46−7% (−) 47 13% (+) 48  37% (++) >90% (+++) −2% (−) Compound activityranges presented are defined as follows: (−−) is less than−10%inhibition; (−) is between −10% to 10% inhibition; (+) is between10% to 30% inhibition; (++) is between 30% to 60% inhibition; and (+++)is greater than 60% inhibition.

Results of Thioflavin T assays of compound effects on aggregation.Compound activity ranges are defined below Table 4. Tested compoundsdemonstrated a variety of effects in the Thioflavin T assays ofaggregation of α-synuclein, CsgA-seeded α-synuclein, and CsgA. Compoundsof Table 4 demonstrated a range of activities. The inhibition of thevarious types of aggregation as described in Table 4 suggests that thecompounds of the invention (e.g., compounds shown in Table 4) may beuseful in preventing α-synuclein aggregation, the seeding of α-synucleinaggregation by CsgA or other microbial amyloids, and the formation ofmicrobial amyloids that may seed α-synuclein aggregation in vivo, andthese compounds may thereby be useful in preventing or treatingParkinson's Disease and other α-synucleinopathies. Oral administrationof these compounds may allow relatively high concentrations to beachieved in the gut, where microbes producing amyloids may be abundant,and the compounds could inhibit their seeding of α-synucleinaggregation. Inhibition by these compounds of aggregation of α-synucleinon its own could be independently beneficial or may be synergistic withtheir inhibition of microbial amyloid-seeded α-synuclein aggregation. Inkeeping with Braak's hypothesis of prion-like propagation of α-synucleinfrom the enteric nervous system to the central nervous system (see,e.g., Rietdijk et al., “Exploring Braak's Hypothesis of Parkinson'sDisease.” Front. Neurol., 13 Feb. 2017), these inhibitory effects couldbe beneficial in preventing propagation of α-synuclein aggregates inboth the enteric and central nervous systems; furthermore, if orallyadministered compounds alleviate a continual seeding of α-synucleinaggregation by microbial amyloids or independent formation ofα-synuclein aggregates, the processes by which subjects may clearα-synuclein aggregates may be able to have a greater net effect (i.e.,the processed may be able to keep pace with the aggregates formed), andthe compounds may thereby be efficacious in preventing or treatingParkinson's Disease and other microbial amyloid-seededα-synucleinopathies. Without being limited by theory, oraladministration may provide particular benefit in the gastrointestinaltract, potentially restoring gastrointestinal function in those patientsin whom it is compromised or in preventing or slowing additional loss ofgastrointestinal function and/or improving one or more symptoms of,e.g., dysphagia, reduced gut motility, gastroparesis, constipation(including chronic constipation and chronic idiopathic constipation),small intestine bacterial overgrowth (SIBO), diarrhea, abdominal painand/or cramping, bloating, flatulence, nausea, or any other symptoms ofirritable bowel syndrome (IBS), inflammatory bowel disease (IBD),ulcerative colitis. Crohn's disease, intestinal hyperpermeability, orany combinations thereof, in patients with α-synucleinopathies or insubjects at risk for developing α-synucleinopathies.

As shown in Table 4, some compounds demonstrated inhibition of only oneor two types of aggregation in the assays, while other compoundsappeared inactive or enhanced one or more types of aggregation, and somecompounds inhibit three types of aggregation in the assays.

Example 33

Enteroendocrine cells (STC1 cell line cells) were treated with E. coliK12 or the ΔcsgBAC, curli-deficient strain at an MOI of 10:1 for 4hours. Cells were lysed, and protein samples assessed by SDS-PAGE andwestern blot for alpha-synuclein (αSyn) and actin, as loading control.The western blot is shown in FIG. 9 . These results indicate that CsgAaffects αSyn aggregation in the gut, for example in enteroendocrinecells.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to plural as isappropriate to the context and/or application. The varioussingular/plural permutations can be expressly set forth herein for sakeof clarity.

Example 34 Preparation of2-(3,5-dihydroxybenzamido)-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 3)

Step 1: Synthesis of ethyl2-(3,5-dimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1.0 g,4.4 mmol) was dissolved in toluene and 3,5-trimethoxybenzoyl chloride(0.89 g, 4.4 mmol) was added. The reaction mixture was stirred for 30min then heated at 110° C. for 3 hrs. The reaction mixture was cooled at0° C. The solid was collected by filtration and washed with cold toluene(2×10 mL) to give ethyl2-(3,5-dimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylateas an off white solid (1.5 g, 86.8%). Preparative High Pressure LiquidChromatography (HPLC) Method H: Shimadzu LC-20AP, column X-Bridge C18(250×19) mm, 5 micron, flow rate 15.0 mL/min, mobile phase A: 5 mMammonium acetate in water, B: 100% acetonitrile. Liquid ChromatographyMass Spectrum (LCMS): 99.67% (RT: 2.412, 258 nm) (MS: ESI +ve 390.5[M+H]).

Step 2: Synthesis of2-(3,5-dimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 3)

Ethyl2-(3,5-dimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(0.20 g, 0.513 mmol) was dissolved in toluene (8.0 mL), The reactionmixture was cooled to 0° C. and a 2 M solution of trimethylaluminum intoluene (0.487 mL, 0.975 mmol) was added. After 30 min,3,4,5-trimethoxyaniline (0.112 g, 0.616 mmol) was added and the mixturewas heated at 110° C. for 4 hrs. After cooling to room temperature, thereaction was quenched with ice water (30 mL) and extracted withdichloromethane (3×30 mL). The organic layer was dried over sodiumsulphate and concentrated. The crude residue was triturated withmethanol (10 mL) to give2-(3,5-dimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 3) (79 mg, 29% yield) as an off white solid. LCMS: 100% (RT:2.286, 254 nm) (MS: ESI +ve 526.83 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δppm: 1.970 (t, 2H, J=26.5), 2.811 (t, 2H, J=24.3) 2.939 (t, 2H J=24.3)3.901 (s, 9H), 3.942 (s, 6H), 6.683 (s, 1H), 6.888 (s, 2H) 7.617 (s,1H), 12.949 (s, 1H).

Example 35 Preparation ofN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide. (Compound 9)

Step 1: Synthesis of ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

A mixture of ethyl2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (10 g, 0.044mol) and 3,4,5-trimethoxybenzoyl chloride (10.23 g, 0.0443 mol) intoluene was stirred for 30 min then heated to 110° C. for 3 hrs. Thereaction mixture was cooled at 0° C. The resulting solid was collectedby filtration and washed with cold toluene (2×50 mL) to obtain ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(10.5 g, 56.4% yield) as an off white solid. LCMS: 94.26% (RT: 2.345,265 nm) (MS: ESI +ve 420.52 [M+H]).

Step 2: Synthesis ofN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide. (Compound 9)

Ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(0.3 g, 0.715 mmol) was dissolved in toluene (10 mL). The reactionmixture was cooled to 0° C. and a 2 M solution of trimethylaluminum intoluene (0.7 mL, 1.4 mmol) was added dropwise and stirring continued for30 min. Aniline (0.086 g, 0.88 mmol) was added and the mixture washeated at 110° C. for 4 hrs. After cooling to room temperature, thereaction mixture was quenched with ice water (40 mL) and extracted withethyl acetate (3×35 mL). The organic layer was dried over sodiumsulphate and concentrated. The crude residue was triturated withmethanol (10 mL) then further purified by reverse phase preparative HPLCto giveN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 9) (17 mg, 5.1% yield). Preparative HPLC Method B. LCMS:98.49% (RT: 2.169, 340.0 nm) (MS: ESI +ve 467.2 [M+H]). ¹H NMR (400 MHz,DMSO-d₆) δ ppm: 1.796 (m, 4H) 2.688 (t, 4H, J=22.42), 3.709 (s, 3H)3.924 (s, 6H) 7.073 (s, 1H), 7.157 (s, 2H), 7.341-7.303 (t, 2H),7.741-7.721 (d, 2H, J=8), 9.871 (s, 1H), 11.273 (s, 1H).

Example 36 Preparation ofN-methyl-2-(3,4,5-trimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 7)

Prepared from ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(300 mg, 0.715 mmol) and 3,4,5-trimethoxy-N-methylaniline (169 mg, 0.858mmol) by a method similar to that described forN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide (Compound 9) to giveN-methyl-2-(3,4,5-trimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 7) (31 mg, 7.60% yield) as an off white solid. PreparativeHPLC Method C. LCMS: 100% (RT: 1.720, 254.0 nm) (MS: ESI +ve 571.4[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.728 (m, 4H), 2.516-2.684 (t,4H, J=33.4), 3.355 (s, 9H) 3.548 (s, 3H) 3.739 (s, 3H) 3.879 (s, 6H),6.134 (s, 2H), 7.298 (s, 2H), 10.511 (s, 1H).

Example 37 Preparation ofN-(3,5-dimethoxyphenyl)-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 5)

Prepared from ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(0.20 g, 0.476 mmol) and 3,5-dimethoxyaniline (0.112 g, 0.572 mmol) by amethod similar to that described forN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide (Compound 9) to giveN-(3,5-dimethoxyphenyl)-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 5) (128 mg, 51% yield) as an off white solid. LCMS: 100% (RT:2.223, 225 nm) (MS: ESI −ve 525.5 [M−H]). ¹H NMR: (400 MHz, DMSO-d₆) δppm: 1.963 (m, 4H), 2.806 (t, 2H, J=24.6), 2.932 (t, 2H, J=24.6), 3.854(s, 6H), 3.938 (s, 3H), 3.978 (s, 6H) 6.341-6.336 (d, 1H, J=2.0),6.879-6.873 (d, 1H, J=2.4), 7.333-7.304 (d, 1H, J=2.9), 7.698 (s, 1H),13.102 (s, 1H).

Example 38 Preparation of2-(3,4,5-trimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 1)

Prepared from ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(0.2 g, 0.477 mmol) and 3,4,5-trimethoxyaniline (0.104 g, 0.572 mmol) bya method similar to that described forN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide (Compound 9) to give2-(3,4,5-trimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 1) (51 mg, 19.22%). LCMS: 98.99% (RT: 2.056, 275.0 nm) (MS:ESI +ve 557.6 [M+H]). ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 1.806 (m, 4H)2.698 (t, 4H J=23.42) 3.642 (s, 3H) 3.736 (s, 15H) 7.161 (s, 2H) 7.207(s, 2H) 9.713 (s, 1H) 11.372 (s, 1H)

Example 39 Preparation of2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 12)

Prepared from ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3carboxylate (100 mg, 0.238 mmol) and ammonium carbonate (45 mg, 0.364mmol) by a method similar to that described forN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide (Compound 9) to give2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 12) (40 mg, 42.97% yield) as an off white solid. LCMS: 100.0%(RT: 1.692, 222 nm) (MS: ESI +ve 391.50 [M+H]). ¹H NMR: (400 MHz,DMSO-d₆) 1.767 (s, 4H), 2.675-2.764 (m, 4H), 3.764 (s, 3H), 3.885 (s,6H), 7.199 (s, 2H), 7.829 (s, 2H) 13.155 (s, 1H)

Example 40 Preparation of2-benzamido-N-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 10)

Step 1: Synthesis of Ethyl2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

Pyridine (1.25 mL) and benzoic acid (542 mg, 4.438 mmol) were added to asolution of ethyl2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (500 mg, 2.219mmol) in dichloromethane (10 mL) at 0° C. After 10 min, phosphorus(V)oxychloride, (1.25 mL) was added. The mixture was allowed to slowly warmto room temperature and stirring continued for 16 h. Ice water (50 mL)was added and the crude mixture was extracted with dichloromethane (3×50mL). The organic layer was dried over sodium sulphate and concentrated.The crude residue was purified by column chromatography eluting withethyl acetate:hexane to give ethyl2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (200 mg,27.4% yield) as off white solid. LCMS: 57.85% (RT: 2.427, 230.0 nm) (MS:ESI +ve 330.08 [M+H])

Step 2: Synthesis of2-benzamido-N-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 10)

Ethyl 2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (100mg, 0.303 mmol) was dissolved in toluene (3.0 mL), The reaction mixturewas cooled to 0° C. and a 2 M solution of trimethylaluminum in toluene(0.3 mL) was added dropwise and stirring continued for 30 min. Aniline(33 mg, 0.364 mmol) was added and the mixture was heated at 110° C. for4 hrs. After cooling to room temperature, the reaction mixture wasquenched with ice water (30 mL) and extracted with dichloromethane (3×30mL). The organic layer was dried over sodium sulphate and concentrated.The crude residue was purified by column chromatography eluting withethyl acetate:hexane then further purified by reverse phase preparativeHPLC to give2-benzamido-N-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 10) (7 mg, 6.1% yield) as an off white solid. LCMS: 100% (RT:2.212, 202.0 nm) (MS: ESI +ve 377.1 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δppm: 1.800 (m, 4H), 2.682-2.887 (t, 4H), 7.10 (s, 1H), 7.332-7.368 (1,2H, J=7.2), 7.558 (m, 3H), 7.702-7.722 (t, 2H, J=8), 7.901 (s, 2H),9.689 (s, 1H), 11.627 (s, 1H).

Example 41 Preparation of2-benzamido-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (Compound 11)

Prepared from ethyl2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (100 mg,0.303 mmol and 3,4,5-trimethoxyaniline (67 mg, 0.364 mmol)) by a methodsimilar to that described forN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide (Compound 9) to give2-benzamido-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 11) (25 mg, 17.7% yield) as an off white solid. PreparativeHPLC Method D. LCMS: 97.97% (RT: 2.082, 202.0 nm) (MS: ESI +ve 467.2[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.767 (m, 4H), 2.711-2.743 (t,4H), 3.649 (s, 3H), 3.764 (s, 6H), 7.124 (s, 2H), 7.586 (m, 3H),7.899-7.917 (d, 2H, J=7.2), 9.635 (s, 1H), 11.499 (s, 1H).

Example 42 Preparation of2-(3,4,5-trimethoxy-N-methylbenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 13)

Step 1: Synthesis of ethyl2-(methylamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

A suspension of ethyl2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5 g, 22.19mmol) and K₂CO₃ (4.6 g, 33.28 mmol) in dimethylformamide (30 mL) wasstirred for 30 min at 0° C. Methyl iodide (1.53 mL, 24.40 mmol) wasadded and reaction mixture was warmed to room temperature and stirredfor 16 h. The reaction mixture was quenched with water (200 mL),extracted with ethyl acetate (3×100 mL), dried over sodium sulfate andconcentrated under reduce pressure. The crude residue was purified byflash chromatography eluting with ethyl acetate:hexane to give ethyl2-(methylamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1.2g, 22.6% yield) as brown solid. LCMS: 88.12%, (RT: 2.139) (MS: ESI +ve240.2 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.24 (t, 3H, J=0.4),1.68 (s, 4H), 2.63 (d, 2H, J=6), 2.86 (s, 3H), 4.12-4.19 (m, 2H), 7.63(s, 1H).

Step 2: Synthesis of ethyl2-(3,4,5-trimethoxy-N-methylbenzamido)-4,5,6,7 tetrahydrobenzo[b]thiophene-3-carboxylate

A mixture of ethyl2-(methylamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1.0g, 4.18 mmol) and 3,4,5-trimethoxybenzoyl chloride (1.93 g, 8.36 mmol)in benzene (20 mL) was heated at 90° C. for 3 h. The reaction mixturewas quenched with water (200 mL), extracted with ethyl acetate (2×100mL), dried over sodium sulfate and concentrated under reduce pressure.The crude residue was purified by flash chromatography eluting withethyl acetate/hexane to give ethyl2-(3,4,5-trimethoxy-N-methylbenzamido)-4,5,6,7tetrahydrobenzo[b]thiophene-3-carboxylate (1.1 g, 60.7% yield) as yellowsolid. LCMS: 80.52% (RT: 1.885) (MS: ESI +ve 434.4 [M+H]).

Step 3:2-(3,4,5-trimethoxy-N-methylbenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 13)

A 2 M solution of trimethylaluminum in toluene (2 mL, 4.0 mmol) wasadded dropwise to a solution of ethyl2-(3,4,5-trimethoxy-N-methylbenzamido)-4,5,6,7tetrahydrobenzo[b]thiophene-3-carboxylat (0.5 g, 1.15 mmol) in toluene(5 mL) at 0° C. The reaction mixture was stirred for 30 min.3,4,5-trimethoxyaniline (0.317 g, 1.73 mmol) was added and the mixturewas heated at 110° C. for 16 h. The reaction mixture was quenched withice water (25 mL), extracted with ethyl acetate (2×25 mL), dried oversodium sulfate and concentrated under reduce pressure. The crude residuewas purified by preparative HPLC to give2-(3,4,5-trimethoxy-N-methylbenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 13) (0.05 g, 7.8% yield) as an off white solid. PreparativeHPLC Method C. LCMS: 100%. (RT: 1.738) (MS: ESI +ve 571.5 [M+H]). ¹HNMR: (400 MHz, DMSO-d₆) δ ppm: 1.75 (s, 5H), 2.64 (s, 3H), 3.18 (s, 3H),3.45 (s, 6H,), 3.63-3.66 (d, 6H, J=14.8), 3.74 (s, 6H), 6.74 (s, 2H),7.03 (s, 2H).

Example 43 Preparation of2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid. (Compound 17)

Step 1: Synthesis of2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid. (Compound 17)

A solution of LiOH (599.6 mg, 14.290 mmol) in water (2 mL) was added toa solution of ethyl2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate(1.0 g, 2.383 mmol) in EtOH (24 mL). The mixture was stirred for 30 minthen heated at 60° C. for 16 h. The reaction mixture was concentratedand diluted with ice water. 1N hydrochloric acid (5 mL) was added toadjust to pH 2 and stirring was continued for 30 min. The resulting pptwas collected by filtration and dried to give 970 mg crude of product.100 mg of crude product was further purified by reverse phasepreparative HPLC to give2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid (Compound 17) (21.75 mg, 23.3% yield) as an off white solidPreparative HPLC Method E: Shimadzu LC-20AP, column Kromasil EternityXT-5 C18 (250×21.2) mm, 5 micron, flow rate 14.0 mL/min, mobile phase A:0.1% trifluoroacetic acid in water and B: 100% acetonitrile. LCMS:100.00% (RT: 1.914, 265.0 nm) (MS: ESI −ve 390.41 [M−H]). ¹H NMR: (400MHz, DMSO-d₆) δ ppm: 1.739-1.749 (m, 4H), 2.649-2.751 (m, 4H), 3.760 (s,3H), 3.883 (s, 6H), 7.210 (s, 2H), 12.459 (s, 1H), 13.450 (s, 1H).

Example 44 Preparation of2-amino-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 14)

Step 1: Synthesis of2-amino-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 14)

Step-1: 2 M trimethylaluminum in toluene (1.32 mL, 2.663 mmol) was addedto a solution of 3,4,5-trimethoxyaniline (363.9 mg, 2.00 mmol) at 0° C.followed by ethyl2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (300 mg, 1.331mmol). The reaction mixture was stirred for 30 min then heated at 75° C.for 16 h. The mixture was quenched in ice water (30 mL), extracted withdichloromethane (3×30 mL), dried over sodium sulphate and concentrated.The crude residue was purified by column chromatography eluting withmethanol:dichloromethane then further purified by reverse phasepreparative HPLC to give2-amino-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 14) (10 mg, 2.1% yield) as an off white solid. PreparativeHPLC Method D. LCMS: 98.26% (RT: 1.770, 230.0 nm) (MS: ESI +ve 363.13[M+1]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.701-1.766 (m, 4H),2.516-2.647 (m, 4H), 3.622-3.671 (s, 3H), 3.744-3.780 (s, 6H), 6.555 (s,2H), 7.059 (s, 2H), 8.843 (s, 1H).

Example 45 Preparation of2-(3,5-dihydroxybenzamido)-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 4)

Step 1. Synthesis of2-(3,5-dihydroxybenzamido)-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 4)

A mixture of2-(3,5-dimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 3) (100 mg, 0.189 mmol), dichloromethane (10 mL) and pentane(12 mL) was cooled at 0° C. Boron tribromide (1 M in dichloromethane)(2.27 mL, 2.278 mmol) was added dropwise and reaction mixture was warmedto room temperature and stirred for 16 h. After cooling to 0° C.,methanol (3 mL) was added dropwise. The reaction mixture wasconcentrated and the crude residue was purified by reverse phasepreparative HPLC to give2-(3,5-dihydroxybenzamido)-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 4) (17 mg, 19.6% yield) as an off white solid. PreparativeHPLC Method A: Shimadzu LC-20AP; column: YMC Actus Triart C18 (250×20)mm, 5 micron, flow rate 16.0 mL/min, mobile phase A: 0.1%trifluoroacetic acid in water, B: 100% acetonitrile. LCMS: 100.00% (RT:1.619, 202.0 nm) (MS: ESI −ve 455.5 [M−H]). ¹H NMR: (400 MHz, DMSO-d₆) δppm: 1.748-1.786 (m, 4H), 2.337-2.755 (t, 4H, J=25.7), 6.449 (s, 1H),6.702-6.723 (s, 3H), 7.951 (s, 1H), 8.899 (s, 2H), 9.070 (s, 1H), 9.780(s, 2H), 11.674 (s, 1H).

Example 46 Preparation of N-phenyl-2-(3,4,5-trihydroxybenzamido)-4,5,6,7tetrahydrobenzo [b] thiophene-3-carboxamide. (Compound 8)

Prepared fromN-phenyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (Compound 9) (100 mg, 0.214 mmol) by asimilar method to that described for (Compound 4) to giveN-phenyl-2-(3,4,5-trihydroxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 8) (5 mg, 5.50% yield Preparative HPLC Method B: ShimadzuLC-20AP; column YMC Actus Triart C18 (250×20) mm, 5 micron, flow rate15.0 mL/min, mobile phase A: 0.1% formic acid in water, B: 100%acetonitrile. LCMS: 100% (RT: 1.739, 230.0 nm) (MS: ESI +ve 425.1[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.773 (m, 4H) 2.679 (t, 2H,J=23.4) 2.779 (t, 2H, J=23.4), 6.886 (s, 2H) 7.152-7.115 (t, 1H),7.397-7.358 (t, 2H), 7.705-7.686 (d, 2H, J=8), 9.015 (s, 1H), 9.448 (s,2H), 9.502 (s, 1H), 11.505 (s, 1H).

Example 47 Preparation ofN-(3,5-dihydroxyphenyl)-2-(3,4,5-trihydroxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 6)

Prepared fromN-(3,5-dimethoxyphenyl)-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 5) (100 mg, 0.190 mmol) by a similar method to that describedfor (Compound 4) to give5,7-dihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 6) (49 mg, 56.5% yield) as an off white solid. PreparativeHPLC Method C: Shimadzu LC-20AP; column Sunfire C18 (150×19) mm, 5micron, flow rate 15.0 mL/min, mobile phase A: 0.1% formic acid inwater, B: 100% acetonitrile. LCMS: 100% (RT: 1.491, 277.0 nm) (MS: ESI−ve 455.5 [M−H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.752 (m, 4H), 2.681(t, 2H, J=25.6), 2.769 (t, 2H J=25.6), 5.981 (s, 1H) 0.6.664-6.644 (d,1H, J=1.6), 6.874 (s, 2H), 9.220 (s, 3H), 9.251 (s, 2H), 11.480 (s, 1H).

Example 48 Preparation of2-benzamido-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo [b]thiophene-3-carboxamide. (Compound 18)

Prepared from2-benzamido-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 11) (100 mg, 0.214 mmol) by a similar method to that describedfor (Compound 4) to give2-benzamido-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 18) (10 mg, 11% yield) as a yellow solid. Preparative HPLCMethod C. LCMS: 100% (RT: 1.679, 202.0 nm) (MS: ESI −ve 423.5 [M−H]). ¹HNMR: (400 MHz, DMSO-d₆) δ ppm: 1.750-1.792 (m, 4H), 2.690-2.743 (t, 4H),6.724 (s, 2H), 7.583-7.647 (m, 3H), 7.870-7.888 (d, 2H, J=7.2), 7.964(s, 1H), 8.931 (s, 2H), 9.190 (s, 1H), 11.744 (s, 1H).

Example 49 Preparation of2-(3,4,5-trihydroxybenzamido)-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 2)

Prepared from2-(3,4,5trimethoxybenzamido)-N-(3,4,5-trimethoxyphenyl)-4,5,6,7tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 1) (100 mg, 0.179 mmol) by a similar method to that describedfor (Compound 4) to give2-(3,4,5-trihydroxybenzamido)-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 2) (11 mg, 12.96% Preparative HPLC Method D: Shimadzu LC-20AP,column X-Bridge C18 (150×19) mm, 5 micron, flow rate 15.0 mL/min, mobilephase A: 0.1% formic acid in water, B: 100% acetonitrile. LCMS: 100%(RT: 1.459, 232.0 nm) (MS: ESI −ve 471.5 [M−H]). ¹H NMR: (400 MHz,DMSO-d₆) δ ppm: 1.773 (m, 4H) 2.679 (t, 2H, J=23.4) 2.779 (t, 2H,J=23.4) 6.761 (s, 2H) 6.886 (S, 2H) 7.966 (s, 1H) 9.006 (s, 4H) 9.445(s, 2H) 11.632 (s, 1H).

Example 50 Preparation of2-(3,4,5-trihydroxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 15)

Prepared from2-(3,4,5-trihydroxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 12) (50 mg, 0.137 mmol) by a similar method to that describedfor (Compound 4) to give2-(3,4,5-trihydroxybenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 15) (9 mg, 18.83%) as an off white solid. Preparative HPLCMethod C. LCMS: 100.00% (RT: 1.488, 223.0 nm) (MS: ESI +ve 349.26[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.759 (m, 4H), 2.651-2.758 (m,4H), 6.896 (s, 2H), 7.693 (s, 1H), 9.412 (s, 3H), 12.750 (s, 1H).

Example 51 Preparation of2-amino-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide.(Compound 16)

Prepared from2-amino-N-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 14) (80 mg, 0.220 mmol) by a similar method to that describedfor (Compound 4) to give2-amino-N-(3,4,5-trihydroxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide(Compound 16) as an off white solid (8 mg, 11.31% yield). PreparativeHPLC Method C. LCMS: 97.39% (RT: 1.469, 230.0 nm) (MS: ESI −ve 319.31[M−H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.682-1.733 (m, 4H),2.547-2.676 (m, 4H), 6.399 (s, 2H), 6.366-6.735 (s, 2H), 7.787 (s, 1H),8.533 (s, 1H), 8.755 (s, 2H).

Example 52 Preparation of5,6,7-trimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxylicacid (Compound 36)

Step 1. Synthesis of5,6,7-trimethoxy-2H-benzo[d][1,3]oxazine-2,4(1H)-dione

1N sodium hydroxide (31.5 mL) was added to a solution of6-amino-3-hydroxy-2,4-dimethoxybenzoic acid (5 g 22.0 mmol) in toluene(60 mL) at 0° C. followed by triphosgene (14.36 g, 48.411 mmol). Thereaction mixture warmed to room temperature over 1 h. The resulting pptwas collected by filtration and triturated with hexane (3×10 mL) to give5,6,7-trimethoxy-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (5.2 g, 87.6%yield) as an off white solid. LCMS: 91.49% (RT: 1.407, 254 nm) (MS: ESI+ve 254.32 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.715 (s, 3H),3.827 (s, 3H), 3.881 (s, 3H), 6.482 (s, 1H), 11.541 (s, 1H).

Step 2. Synthesis of ethyl5,6,7-trimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-1,4-dihydro quinoline-3-carboxylate

Ethyl 3-oxo-3-(3,4,5-trimethoxyphenyl)propanoate (2.9 g, 10.273 mmol)was dissolved in dry dimethylformamide (40 mL) and cooled to 0° C.Sodium hydride (60% in mineral oil) (1.23 g, 30.819 mmol) was added andthe reaction mixture was stirred for 20 min.5,6,7-trimethoxy-2H-benzo[d][1.3]oxazine-2,4(1H)-dione (5.2 g, 20.55mmol) was added and the mixture was allowed to warm to room temperaturethen heated at 80° C. for 16 h. After cooling to room temperature, icewater (250 mL) was added and the pH was adjusted to 6 by the addition of1N hydrochloric acid (10 mL). The mixture was extracted with ethylacetate (3×100 mL), dried over sodium sulphate and concentrated. Thecrude product was purified by column chromatography eluting withmethanol:dichloromethane to give ethyl5,6,7-trimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxylate(1.7 g, 31.4% yield) as brown liquid. LCMS: 70.75% (RT: 1.517, 230.0 nm)(MS: ESI +ve 474.52 [M+H]). Step 3. Synthesis of5,6,7-trimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxylicacid. (Compound 36)

A mixture of ethyl5,6,7-trimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxylate(700 mg 1.479 mmol) and 2N sodium hydroxide (14 mL) was heated at 90° C.for 48 h. The reaction mixture was cooled to room temperature and the pHwas adjusted to 4 by the addition of 1N hydrochloric acid. The mixturewas extracted with ethyl acetate (3×40 mL), dried over sodium sulphateand concentrated. The crude residue was purified by reverse phasepreparative HPLC to give5,6,7-trimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxylicacid (Compound 36) (26 mg, 4.0% yield). as an off white solid.Preparative HPLC Method F: Shimadzu LC-20AP, column X-Bridge C18(250×19) mm, 5 micron, flow rate 14.0 mL/min, mobile phase A: 5 mMammonium bicarbonate and 0.1% ammonia in water, B: 100% acetonitrile.LCMS: 99.81% (RT=1.908, 265 nm) (MS: ESI +ve 446.0 [M+H]). ¹H NMR: (400MHz, DMSO-d₆) δ ppm: 3.24 (s, 3H), 3.774 (s, 6H), 3.800 (s, 3H), 3.832(s, 3H), 3.891 (s, 3H), 6.710 (s, 2H) 7.057 (s, 1H).

Example 53 Preparation of5,6,7-trimethoxy-4-oxo-N,2-bis(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide. (Compound 40)

Step 1. Synthesis of5,6,7-trimethoxy-4-oxo-N,2-bis(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide. (Compound 40)

(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-h]pyridinium3-oxide hexafluorophosphate (HATU) (0.460 g, 1.212 mmol) was added to asolution of5,6,7-trimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxylicacid (Compound 36) (0.36 g, 0.808 mmol) in dimethylformamide (4 mL), at0° C. After 30 min, 3,4,5-trimethoxy aniline (0.161 g, 0.889 mmol) wasadded. After 15 min. N,N-diisopropylethylamine (0.40 mL, 2.424 mmol) wasadded and the mixture was stirred at room temperature for 16 h. Icewater (40 mL) was added and the mixture was extracted with ethyl acetate(3×40 mL), dried and concentrated. The crude residue was purified byreverse phase preparative HPLC to give5,6,7-trimethoxy-4-oxo-N,2-bis(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 40) (90 mg, 18.24% yield) as an off white solid. PreparativeHPLC Method G: Shimadzu LC-20AP; column Sunfire C18 (250×19) mm, 5micron, flow rate 15.0 mL/min, mobile phase A: 0.1% trifluoroacetic acidin water, B: 100% acetonitrile. LCMS: 99.80% (RT 1.463, 225 nm) (MS: ESI+ve 611.52 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.612-3.888 (m,27H), 6.965-7.215 (m, 5H), 10.477 (s, 1H), 11.572 (s, 1H).

Example 54 Preparation of5,6,7-trihydroxy-4-oxo-N,2-bis(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide. (Compound 44)

Step 1. Synthesis of5,6,7-trihydroxy-4-oxo-N,2-bis(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 44)

Borontribromide (1M in dichloromethane) (0.786 mL, 0.786 mmol) was addeddrop wise to a solution of 5,6,7-trimethoxy-4-oxo-N,2-bis(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 40) (40 mg, 0.0655 mmol) in dichloromethane at −10° C. Thereaction mixture was allowed to slowly warm to room temperature andstirred for 16 h. The reaction mixture was cool to 0° C. and methanol (4mL) was added dropwise. After 20 min, the mixture was concentrated andthe resulting solid was triturated with MTBE (2×4 mL), then pentane (4×4mL). Further purification by reverse phase preparative HPLC gave5,6,7-trihydroxy-4-oxo-N,2-bis(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 44) (2.27 mg, 7.0% yield) as a white solid. Preparative HPLCMethod D. LCMS: 100% (RT 1.557, 202 nm) (MS: ESI +ve 485.0 [M+H]). ¹HNMR: (400 MHz, DMSO-d₆) δ ppm: 6.533-6.601 (m, 5H), 7.900 (s, 2H),8.526-8.830 (s, 4H), 8.894-9.179 (s, 2H), 9.759 (s, 1H) 11.667 (s, 1H)14.439 (s, 1H).

Example 55 Preparation of5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 19)

Step 1: Synthesis of diethyl2-(((3,4,5-trimethoxyphenyl)amino)methylene)malonate

3,4,5-trimethoxy aniline (25 g, 0.136 mol) was dissolved in diethylethoxy methylene malonate (28 mL, 0.140 mol) and the mixture was heatedat 140° C. for 2 hrs. The crude mixture was dissolved in dichloromethane(100 mL), concentrated then triturated with methanol (2×20 mL) to givediethyl 2-(((3,4,5-trimethoxyphenyl)amino)methylene)malonate (43.5 g,99.6% yield) as brown solid. LCMS: 100% (RT: 2.286, 202 nm) (MS: ESI +ve354.0 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 1.22-1.28 (m, 6H),3.63-3.66 (d, 3H, J=13.6), 3.81 (s, 6H), 4.10-4.15 (m, 2H), 4.17-4.23(m, 2H), 6.73 (s, 2H) 8.37-8.40 (d, 1H, J=12) 10.67-10.71 (d, 1H, J=16)

Step 2: Synthesis of ethyl5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate

A solution of diethyl2-(((3,4,5-trimethoxyphenyl)amino)methylene)malonate (5 g, 0.014 mol) indiphenyl ether (25 mL) was heated at 240° C. for 15 min. The reactionmixture was cooled to room temperature and the resulting ppt wascollected by filtration and triturated with hexane (3×10 mL) to giveethyl 5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (1.53 g,35.2% yield) as a brown solid. LCMS: 84.53% (RT: 1.816, 254 nm) (MS: ESI+ve 308 [M+H]).

Step 3: Synthesis of5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

A mixture of ethyl5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (3 g, 0.009mol) in 2N sodium hydroxide (150 mL) was heated at 90° C. for 3 h. Aftercooling to room temperature, the pH was adjusted to 4 by the addition of2N hydrochloric acid. The resulting ppt was collected by filtration andtriturated with methanol (5 mL) to give5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.2 g,80.7% yield) as a pink solid. LCMS: 84.94% (RT: 1.348, 315 nm) (MS: ESI+ve 280.0 [M+H]).

Step 4: Synthesis of5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 19)

5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (850 mg,3.043 mmol) was dissolved in dimethylformamide (5 mL) and cooled to 0°C. HATU (1.7 g, 4.565 mmol) followed by 3,4,5-trimethoxy aniline (557.6mg, 3.043 mmol) after 30 min. N,N-Diisopropylethylamine (1.56 mL, 9.145mmol) was added and the mixture was allowed to slowly warm to roomtemperature and stirred for 16 h. The reaction mixture was quenched intoice water (50 mL), extracted with dichloromethane (3×50 mL), dried oversodium sulphate and concentrated. The crude residue was purified bycolumn chromatography eluting with methanol:dichloromethane to give 350mg which was further purified by reverse phase preparative HPLC to give5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) (110 mg, 8.13% yield) as an off white solid. PreparativeHPLC Method H: Shimadzu LC-20AP, column X-Bridge C18 (250×19) mm, 5micron, flow rate 15.0 mL/min, mobile phase A: 5 mM ammonium acetate inwater. B: 100% acetonitrile. LCMS: 99.86% (RT: 1.480, 254 nm) (MS: ESI+ve 445.4 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.65 (s, 3H), 3.79(s, 3H), 3.81 (s, 6H), 3.86 (s, 3H), 3.92 (S, 3H), 6.97 (s, 1H), 7.11(s, 2H), 8.71 (s, 1H), 12.74 (s, 1H).

Example 56 Preparation of5,7-dimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 21)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(0.99 g, 3.97 mmol) and 3,4,5-trimethoxy aniline (0.73 g, 3.99 mmol) bya procedure similar to that described for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 19) to give5,7-dimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 21) (612 mg, 37.0% yield) as an off white solid. PreparativeHPLC Method H: LCMS: 100% (RT: 1.459, 235.0 nm) (MS: ESI +ve 415.3[M+H]). ¹H NMR: (400 MHz, CDCl₃) δ ppm: 3.749 (s, 3H), 3.862 (s, 9H),4.066 (s, 3H), 6.441 (s, 2H), 7.109 (s, 1H), 7.543 (s, 1H), 8.983 (s,1H), 12.640 (s, 1H)

Example 57 Preparation of5,7-dimethoxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide.(Compound 25)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(300 mg, 1.20 mmol) and aniline (0.11 mL, 1.20 mmol) by a proceduresimilar to that described for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 19) to give5,7-dimethoxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide(Compound 25) (40 mg, 10.3% yield) as an off white solid. PreparativeHPLC Method D. LCMS: 95.73% (RT: 1.466, 260.0 nm) (MS: ESI +ve 325.43[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.87 (s, 3H), 3.89 (s, 3H),6.52 (d, 1H, J=2) 6.66-6.67 (d, 1H, J=2) 7.06-7.10 (t, 1H, J=7.2),7.34-7.38 (t, 2H, J=8), 7.72-7.74 (d, 2H, J=7.6), 8.64-8.66 (d, 1H,J=6.4), 12.41-12.43 (d, 1H, J=6.8), 12.70 (s, 1H).

Example 58 Preparation ofN-(3-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 26)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(300 mg, 1.20 mmol) and 3-aminophenol (131.36, 1.20 mmol) by a proceduresimilar to that described for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 19) to giveN-(3-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 26) (18 mg, 4.4% yield) as an off white solid. PreparativeHPLC Method F. LCMS: 100.00% (RT: 1.399, 202 nm) (MS: ESI +ve 341.27[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) (73043) δ ppm: 3.34 (s, 3H), 3.87 (s,3H), 6.46-6.51 (t, 2H, J=9.2), 6.66 (s, 1H), 6.99-7.01 (d, 1H, J=8)7.10-7.14 (t, 1H, J=8.2), 7.34 (s, 1H), 8.62 (s, 1H) 9.43 (s, 1H)12.40-12.39 (d, 1H, J=4.8), 12.60 (s, 1H).

Example 59 Preparation of N-(3-hydroxyphenyl)-5, 6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide. (Compound 28)

Prepared from 5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (500 mg, 1.79 mol) and 3-aminophenol (195.3 mg, 1.79 mmol) by aprocedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to give N-(3-hydroxyphenyl)-5, 6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (Compound 28) (15mg, 2.3% yield) as a white solid. Preparative HPLC Method D. LCMS:99.36% (RT: 1.419, 317.0 nm) (MS: ESI +ve 371.47 [M+H]). ¹H NMR: (400MHz, DMSO-d₆) δ ppm: 3.784 (s, 3H), 3.844 (s, 3H), 3.922 (s, 3H),6.469-6.493 (d, 1H, J=9.6), 7.008 (m, 2H), 7.125 (m, 1H), 7.325 (s, 1H),8.693 (s, 1H), 9.444 (s, 1H), 12.546 (s, 1H), 12.626 (s, 1H).

Example 60 Preparation of5,6,7-trimethoxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide.(Compound 30)

Prepared from 5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (200 mg, 0.716 mmol) and aniline (66.6 mg, 0.716 mmol) by aprocedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to give5,6,7-trimethoxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide(Compound 30) (43.5 mg, 16.9% yield) as an off white solid. PreparativeHPLC Method C. LCMS: 100.00% (RT: 1.520, 202.0 nm) (ESI +ve (M+1)355.48). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.801 (s, 3H), 3.866 (s, 3H),3.937 (s, 3H), 7.005 (s, 1H), 7.095 (m, 1H), 7.371 (m, 2H), 7.723-7.743(d, 2H, J=8), 8.720 (s, 1H), 12.569 (s, 1H), 12.698 (s, 1H).

Example 61 Preparation ofN-(4-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 29)

Prepared from 5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (300 mg, 1.075 mmol) and 4-aminophenol (117 mg, 1.075 mmol) by aprocedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to giveN-(4-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 29) (34 mg, 8.55%) as a white solid. Preparative HPLC MethodD. LCMS: 100.00% (RT: 1.393, 254.0 nm) (MS: ESI +ve 371.4 [M+H]). ¹HNMR: (400 MHz, DMSO-d₆) δ ppm: 3.782 (s, 3H), 3.840 (s, 3H), 3.970 (s,3H), 6.586 (s, 2H), 6.734-6.756 (d, 2H, J=8.8), 6.985 (s, 1H)7.496-7.518 (d, 2H, J=9.2), 8.672 (s, 1H), 9.241 (s, 1H), 12.415 (s,1H).

Example 62 Preparation ofN-(4-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 31)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(300 mg, 1.20 mmol) and 4-aminophenol (131.36, 1.20 mmol) by a proceduresimilar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to giveN-(4-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 31) (7.2 mg, 1.8% yield) as an off white solid. PreparativeHPLC Method D. LCMS: 98.99% (RT: 1.365, 259 nm) (MS: ESI +ve 341.27[M+H]). ¹H NMR: (400 MHz, DMSO-de) δ ppm: 3.85 (s, 3H), 3.87 (s, 3H),6.48-6.49 (d, 1H, J=2), 6.64-6.65 (d, 1H, J=2), 6.72-6.74 (d, 2H, J=8.8)7.49-7.52 (d, 2H, J=8.8) 8.60 (s, 1H), 9.21 (s, 1H) 12.43 (s, 2H).

Example 63 Preparation ofN-(3-fluoro-4-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 34)

Prepared from 5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (300 mg, 1.075 mmol) and 5-amino-2-fluorophenol (136 mg, 1.075mmol) by a procedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to giveN-(3-fluoro-4-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 34) (34 mg, 8.2% yield) as an off white solid. PreparativeHPLC Method I: Shimadzu LC-20AP; column Sunfire C18 (150×19) mm, 5micron, flow rate 15.0 mL/min, mobile phase A: 0.1% formic acid inwater, B: acetonitrile:methanol (1:1). LCMS: 100.00% (RT: 1.414, 254.0nm) (MS: ESI +ve 389.4 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.783(s, 3H), 3.840 (s, 3H), 3.922 (s, 3H), 6.901-6.947 (m, 1H), 6.986 (s,1H), 7.118-7.140 (d, 1H, J=8.8), 7.762-7.801 (dd, 1H, J=2.4.15.5) 8.683(s, 1H), 9.623 (s, 1H) 12.430 (s, 1H), 12.575 (s, 1H).

Example 64 Preparation of5,7-dimethoxy-N-methyl-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 35)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(300 mg, 1.20 mmol) and 3,4,5-trimethoxy-N-methylaniline (237 mg, 1.20mmol) by a procedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to give5,7-dimethoxy-N-methyl-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 35) (40 mg, 7.8% yield) as an off white solid. PreparativeHPLC Method D. LCMS: 100.00% (RT: 1.344, 225 nm) (MS: ESI +ve 429.39[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.279 (s, 3H), 3.569 (s, 3H),3.661 (s, 6H), 3.743 (s, 3H), 3.795 (s, 3H), 6.301 (s, 1H), 6.390 (s,1H), 6.640 (s, 2H), 7.622 (s, 1H), 11.347 (s, 1H).

Example 65 Preparation ofN-(3-fluoro-4-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 37)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(300 mg, 1.20 mmol) and 4-amino-2-fluorophenol (152 mg, 1.20 mmol) by aprocedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to giveN-(3-fluoro-4-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 37) (26 mg, 6.0% yield) as a white solid. Preparative HPLCMethod A. LCMS: 100.0% (RT: 1.409, 260.0 nm) (MS: ESI +ve 359.08 [M+H]).¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.865 (s, 6H), 6.513-6.518 (d, 1H,J=2), 6.659-6.664 (d, 1H, J=2), 6.900-6.946 (t, 1H, J=9.2), 7.147-7.168(d, 1H, J=8.4), 7.764-7.804 (q, 1H), 8.617-8.634 (d, 1H, J=6.8), 9.621(s, 1H), 12.398-12.414 (d, 1H, J=6.4), 12.576 (s, 1H).

Example 66 Preparation of N-(2-hydroxyphenyl)-5, 6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide. (Compound 39)

Prepared from 5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (500 mg, 1.79 mmol) and 2-aminophenol (195.3 mg, 1.79 mmol) by aprocedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to giveN-(2-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 39) (40 mg, 6.0% yield) as a brown solid. Preparative HPLCMethod B. LCMS: 98.57% (RT: 3.584, 254.0 nm) (MS: ESI +ve 371.4 [M+H]).¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.780-3.835 (s, 6H), 3.885-3.919 (s,3H), 6.763-6.974 (m, 3H), 8.301-8.321 (d, 1H, J=8), 8.685

Example 67 Preparation ofN-(2-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 41)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(300 mg, 1.20 mmol) and 2-aminophenol (131.3 mg, 1.20 mmol)) by aprocedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to giveN-(3-fluoro-4-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 41) (4.0 mg, 1.0% yield) as a white solid. Preparative HPLCMethod B. LCMS: 100.0% (RT: 1.477, 260.0 nm) (MS: ESI +ve 341.33 [M+H]).¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.878 (s, 6H), 6.492-6.496 (d, 1H,J=1.6), 6.657-6.662 (d, 1H, J=2), 6.765-6.807 (m, 1H), 6.880-6.912 (t,2H, J=6.4), 8.313-8.333 (d, 1H, J=8), 8.634 (s, 1H), 10.008 (s, 1H),12.623 (s, 1H).

Example 68 Preparation ofN-(3,5-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 49)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(0.15 g, 0.602 mmol) and. 3,5-dimethoxy aniline (0.091 g, 0.602 mmol) bya procedure similar to that reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to giveN-(3,5-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 49) as an off white solid. Prep HPLC Method C. LCMS: 100% (RT1.532, 260 nm) (MS: ESI +ve 385.41 [M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δppm: 3.776 (s, 6H), 3.885-3.870 (d, 6H, J=6), 6.240 (s, 1H), 6.523-6.518(d, 1H J=2), 6.665-6.661 (s, 1H J=1.6), 6.968-6.963 (d, 2H, J=2) 8.648(s, 1H) 12.499 (s, 1H), 12.695 (s, 1H).

Example 69 Preparation of4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide. (Compound 45)

Prepared from 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (150 mg,0.7932 mmol) and aniline (73 mg, 0.7932 mmol) by a procedure similar tothat reported for5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) to give 4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide(Compound 45) (80.5 mg, 38.2% yield). Preparative HPLC Method C. LCMS:100.00% (RT: 2.087, 226 nm) (MS: ESI +ve 265.3 [M+H]). ¹H NMR: (400 MHz,DMSO-d₆) δ ppm: 7.092-7.128 (t, 1H, J=7.2) 7.366-7.405 (t, 2H, J=7.8),7.540-7.577 (t, 1H, J=7.4), 7.745-7.854 (m, 4H), 8.340-8.358 (d, 1H,J=7.2), 8.907 (s, 1H), 12.52 (s, 1H), 12.98 (s, 1H).

Example 70 Preparation ofN-(3-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide. (Compound46)

Step 1. Synthesis ofN-(3-(benzyloxy)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide

A mixture of 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (150 mg,0.7932 mmol) and HATU (452 mg, 1.1898 mmol) in dimethylformamide (10 mL)was cooled to 0° C. After 30 min, 3-(benzyloxy)aniline (158 mg, 0.7932mmol) was added followed by N,N-diisopropylethylamine (0.75 mL, 3.966mmol). The mixture slowly warmed to room temperature and was stirred for16 h. The reaction mixture was quenched into ice water (25 mL),extracted with ethyl acetate (3×20 mL), dried over sodium sulphate andconcentrated to giveN-(3-(benzyloxy)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide asbrown solid (250 mg, 85.12%). LCMS: 53.13% (RT: 1.855, 202.0 nm) (MS:ESI +ve 371.36 [M+H]).

Step 2. Synthesis ofN-(3-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide. (Compound46)

N-(3-(benzyloxy)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (200mg, 0.54025 mmol) was dissolved in dimethylformamide (15 mL) 10%palladium on carbon (50% moisture) (50 mg) was added and reactionmixture was stirred at room temperature under 2 kg pressure of hydrogengas for 24 h in an autoclave. The catalyst was removed by filtrationthrough a pad of celite and the filtrate concentrated. The crude productwas purified by reverse phase preparative HPLC to giveN-(3-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Compound46) (16.0 mg, 12.6% yield) as an off a white solid. Preparative HPLCMethod D. LCMS: 100.00% (RT: 1.463, 202 nm) (MS: ESI +ve 281.3 [M+H]),¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 6.502-6.522 (d, 1H, J=8), 7.025-7.045(d, 1H, J=8), 7.131-7.171 (t, 1H, J=8), 7.370 (s, 1H), 7.530-7.567 (t,1H, J=7.4), 7.757-7.846 (m, 2H), 8.33-8.353 (d, 1H, J=8), 8.890 (s, 1H),9.492 (s, 1H), 12.46 (s, 1H), 13.0 (s, 1H).

Example 71 Preparation of5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 20)

Step 1. Synthesis of5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 20)

Step 1.5,6,7-trimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 19) (300 mg, 0.675 mmol) was dissolved in a mixture ofdichloromethane (8 mL) and pentane (4 mL) and cooled to 0° C. Borontribromide (1 M in dichloromethane) (8.10 mL, 8.100 mmol) was addeddropwise and the reaction mixture was allowed to slowly warm to roomtemperature and stirred for 16 h. After cooling to 0° C. methanol (8 mL)was added dropwise and the mixture was concentrated. The crude residuewas purified by reverse phase preparative HPLC to give5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) (30 mg, 12.3% yield) as an off white solid. PreparativeHPLC Method A. LCMS: 99.24% (RT: 1.269, 270.0 nm) (MS: ESI −ve 359.4[M−H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 6.605 (s, 1H), 6.709 (s, 2H),7.894 (s, 1H), 8.611 (s, 1H), 8.780 (s, 1H), 8.913 (s, 2H), 10.409 (s,1H), 11.414 (s, 1H), 12.807 (s, 1H), 13.567 (s, 1H).

Example 72 Preparation of Synthesis of5,7-dihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 22)

Prepared from5,7-dimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 21) (250 mg, 0.603 mmol) by a procedure similar to thatdescribed for5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,7-dihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 22) (30 mg, 14.4% yield) as an off white solid. PreparativeHPLC Method A. LCMS: 98.98% (RT: 2.792, 260.0 nm) (MS: ESI +ve 345.5[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 6.204 (s, 1H), 6.482 (s, 1H),6.688 (s, 2H), 7.918 (s, 1H), 8.660 (s, 1H), 8.934 (s, 2H), 10.667 (s,1H), 11.270 (s, 1H), 12.832 (s, 1H), 13.670 (s, 1H).

Example 73 Preparation of5,7-dihydroxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide.(Compound 27)

Prepared from5,7-dimethoxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide(Compound 25) (150 mg, 0.462 mmol) by a procedure similar to thatdescribed for5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,7-dihydroxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide.(Compound 27) (15 mg, 3.3% yield) as an off white solid. PreparativeHPLC Method D. LCMS: 95.66% (RT: 1.609, 272.0 nm) (MS: ESI +ve 297.21[M+H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 6.232 (s, 1H), 6.500 (s, 1H),7.082-7.117 (t, 1H, J=7), 7.345-7.380 (t, 2H, J=7), 7.451 (s, 2H), 8.710(s, 1H), 10.673 (s, 1H), 11.692 (s, 1H), 12.922 (s, 1H), 13.696 (s, 1H).

Example 74 Preparation of5,6,7-trihydroxy-N-(3-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 32)

Prepared fromN-(3-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 28) (150 mg, 0.405 mmol) by a procedure similar to thatdescribed for5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,6,7-trihydroxy-N-(3-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 32) (4 mg, 3.0% yield) as an off white solid. Preparative HPLCMethod D. LCMS: 97.28% (RT: 1.828, 202 nm) (MS: ESI −ve 327.3 [M−H]). ¹HNMR: (400 MHz, DMSO-d₆) δ ppm: 6.478-6.498 (d, 1H, J=8), 6.650 (s, 1H),6.989-7.009 (d, 1H, J=8), 7.102-7.142 (t, 1H J=8), 7.333 (s, 1H), 8.230(s, 1H), 8.625 (s, 1H), 9.472 (s, 1H), 11.744 (s, 1H) 13.537 (s, 1H).

Example 75 Preparation of 5, 6,7-trihydroxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide.(Compound 33)

Prepared from 5, 6,7-trimethoxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide (500 mg,1.412 mmol) (Compound 30) by a procedure similar to that described for5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,6,7-trihydroxy-4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide(Compound 33) (20 mg, 4.5% yield) as an off white solid. PreparativeHPLC Method C. LCMS: 100.00% (RT: 1.481, 229.0 nm) (MS: ESI −ve 311.25[M−H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 6.62 (s, 1H), 7.12-7.08 (t,1H, J=8), 7.39-7.35 (t, 2H, J=8), 7.74-7.72 (d, 2H, J=8), 8.67 (s, 1H),8.83 (s, 1H), 10.50 (s, 1H), 11.81 (s, 1H), 12.90 (s, 1H), 13.54 (s,1H).

Example 76 Preparation of5,6,7-trihydroxy-N-(4-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 38)

Prepared fromN-(4-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 29) (100 mg, 0.270 mmol) by a procedure similar to thatdescribed for5,6,7-trihydoxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,6,7-trihydroxy-N-(4-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 38) (7 mg, 7.9% yield) as an off white solid. Preparative HPLCMethod C. LCMS: 95.95% (RT: 2.907, 278.0 nm) (MS: ESI +ve 329.43 [M+H]).¹H NMR: (400 MHz, DMSO-de) δ ppm: 6.593 (s, 1H), 6.732-6.754 (d, 2HJ=8.8), 7.495-7.516 (d, 2H, J=8.4), 8.618 (s, 1H), 8.795 (s, 1H) 9.272,(s, 1H), 10.409 (s, 1H), 11.529 (s, 1H), 12.839 (s, 1H), 13.574 (s, 1H).

Example 77 Preparation of5,7-dihydroxy-N-methyl-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide.(Compound 42)

Prepared from5,7-dimethoxy-N-methyl-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamide(150 mg, 0.350 mmol) by a procedure similar to that described for5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,7-dihydroxy-N-methyl-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 42) (4 mg, 3.19%) as an off white solid. Preparative HPLCMethod G. LCMS: 100.00% (RT: 1.252, 202 nm) (MS: ESI +ve 359.26 [M+H]).¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 3.197 (s, 3H), 5.995 (s, 1H), 6.186(s, 3H), 7.774 (s, 1H), 8.117 (s, 1H), 8.925 (s, 2H), 10.926 (s, 1H),11.870 (s, 1H), 14.386 (s, 1H).

Example 78 Preparation of5,7-dihydroxy-N-(3-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 48)

Prepared fromN-(3-hydroxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 26) (100 mg, 0.294 mmol) by a procedure similar to thatdescribed for5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,7-dihydroxy-N-(3-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 48) as an off white solid. Preparative HPLC Method G. LCMS:98.54% (RT: 1.464, 263.0 nm) (MS: ESI ve 313.31 [M+H]). ¹H NMR: (400MHz, DMSO-d₆) δ ppm: 6.231 (s, 1H), 6.517-6.509 (d, 2H, J=3.2),7.012-6.992 (d, 1H J=8.0), 7.157-7.137 (d, 1H, J=8.0), 7.137-7.117 (d,1H, J=8.0), 7.340 (s, 1H), 8.721-8.704 (d, 1H, J=6.8), 9.481 (s, 1H),10.704 (s, 1H), 11.587 (s, 1H), 12.920-12.905 (d, 1H, J=6.6), 13.650 (s,1H).

Example 79 Preparation of5,6,7-trihydroxy-N-(2-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 43)

Prepared fromN-(2-hydroxyphenyl)-5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(100 mg, 0.270 mmol) (Compound 39) by a procedure similar to thatdescribed for5,6,7-trihydoxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to give5,6,7-trihydroxy-N-(2-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 43) (7.0 mg, 7.1% yield) as an off white solid. PreparativeHPLC Method D. LCMS: 100.00% (RT: 1.423, 278.0 nm) (MS: ESI −ve 327.4[M−H]). ¹H NMR: (400 MHz, DMSO-d₆) δ ppm: 6.600 (s, 1H), 6.802 (m, 1H),6.903-6.913 (d, 2H, J=4), 8.380-8.400 (d, 1H, J=8), 8.651 (s, 2H), 9.998(s, 2H), 11.823 (s, 1H) 12.800 (s, 1H), 13.752 (s, 1H).

Example 80 Preparation ofN-(3,5-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-1,4-dihydroquinoline-3-carboxamide.(Compound 47)

Prepared fromN-(3,5-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 49) (0.15 mg, 0.39 mmol) by a procedure similar to thatdescribed for5,6,7-trihydroxy-4-oxo-N-(3,4,5-trihydroxyphenyl)-1,4-dihydroquinoline-3-carboxamide(Compound 20) to giveN-(3,5-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 47) (5 mg, 3.9% yield) as an off white solid. Prep HPLC MethodC. LCMS: 98.10% (RT: 1.373, 230.0 nm) (MS: ESI +ve 329.31 [M+H]). ¹HNMR: (400 MHz, DMSO-d₆) δ ppm: 5.96 (s, 1H), 6.20 (s, 1H), 6.49 (s, 1H),6.63 (s, 2H), 8.69 (s, 1H), 9.28 (s, 2H) 10.61 (s, 1H) 11.52 (s, 1H)12.84 (s, 1H) 13.73 (s, 1H).

Example 81 Preparation of 3,4,5-trimethoxyphenyl5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate. (Compound 23)

Step 1: Synthesis of 3,4,5-trimethoxyphenyl5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate. (Compound 23)

A mixture of 5,6,7-Trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (500 mg, 1.790 mmol), N,N-dimethylpyridin-4-amine (218 mg, 1.790mmol) and N,N-dimethylpyridin-4-amine (218 mg, 1.790 mmol) indimethylformamide (25 mL) was cooled to 0° C.(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (1.7 g, 4.476 mmol) was added and reaction the reaction mixturewas stirred for 30 min. 3,4,5-trimethoxyphenol (329.7 mg, 1.790 mmol)was added followed by N,N-diisopropylethylamine (1.2 mL, 7.162 mmol).The mixture slowly warmed to room temperature and was stirred for 16 h.The reaction mixture was quenched into ice water (50 mL) and theresulting ppt was collected by filtration and triturated with methanol.The crude residue was purified by column chromatography eluting withmethanol:dichloromethane to give 140 mg of product which was furtherpurified by reverse phase preparative HPLC to give3,4,5-trimethoxyphenyl5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (Compound 23)(35.8 mg, 4.49%) as white solid. Preparative HPLC Method C. LCMS: 97.85%(RT: 1.402, 259.0 nm) (MS: ESI +ve 446.46 [M+H]). ¹H NMR: (400 MHz,DMSO-d₆) δ ppm: 3.36 (s, 3H), 3.78 (s, 12H), 3.91 (s, 3H), 6.55 (s, 2H),6.94 (s, 1H), 8.60 (s, 1H), 12.10-12.12 (s, 1H).

Example 82 Preparation of 3,4,5-trimethoxyphenyl5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate. (Compound 24)

Prepared from 5,7-dimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid(0.9 g, 3.61 mmol) and 3,4,5-trimethoxyphenol by a procedure similar tothat described for 3,4,5-trimethoxyphenyl5,6,7-trimethoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (Compound 23)give5,7-dimethoxy-4-oxo-N-(3,4,5-trimethoxyphenyl)-1,4-dihydroquinoline-3-carboxamidef (40 mg, 2.7% yield) as off white solid. Preparative HPLC Method D.LCMS: 98.12% (RT: 1.495, 261.0 nm) (MS: ESI +ve 416.49 [M+H]). ¹H NMR:(400 MHz, CDCl₃) (69274) δ ppm: 3.66 (s, 3H) 3.77 (s, 6H) 3.80 (s, 3H)3.91 (s, 3H) 6.44-6.60 (m, 4H), 8.52 (s, 1H) 11.98 (s, 1H).

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (for example, bodiesof the appended claims) are generally intended as “open” terms (forexample, the term “including” should be interpreted as “including butnot limited to,” the term “having” should be interpreted as “having atleast,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.). It will be further understood by those withinthe art that if a specific number of an introduced claim recitation isintended, such an intent will be explicitly recited in the claim, and inthe absence of such recitation no such intent is present. For example,as an aid to understanding, the following appended claims can containusage of the introductory phrases “at least one” and “one or more” tointroduce claim recitations. However, the use of such phrases should notbe construed to imply that the introduction of a claim recitation by theindefinite articles “a” or “an” limits any particular claim containingsuch introduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (for example, “a” and/or “an” should be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould be interpreted to mean at least the recited number (for example,the bare recitation of “two recitations.” without other modifiers, meansat least two recitations, or two or more recitations). Furthermore, inthose instances where a convention analogous to “at least one of A. B,and C, etc.” is used, in general such a construction is intended in thesense one having skill in the art would understand the convention (forexample, “a system having at least one of A. B, and C” would include butnot be limited to systems that have A alone, B alone. C alone. A and Btogether, A and C together. B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (for example. “a system having at least one of A, B, orC” would include but not be limited to systems that have A alone, Balone, C alone, A and B together. A and C together. B and C together,and/or A, B, and C together, etc.). It will be further understood bythose within the art that virtually any disjunctive word and/or phrasepresenting two or more alternative terms, whether in the description,claims, or drawings, should be understood to contemplate thepossibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than.” “less than.” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

Wherever a method of using a composition (e.g., a method comprisingadministering a composition to a subject having an amyloid disorder) isdisclosed herein, the corresponding composition for use is alsoexpressly contemplated. For example, for the disclosure of a method ofinhibiting, ameliorating, reducing the likelihood, delaying the onsetof, treating, or preventing an amyloid disorder administering acomposition to a subject, the corresponding composition for use ininhibiting, ameliorating, reducing the likelihood, delaying the onsetof, treating, or preventing the amyloid disorder is also expresslycontemplated.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1-77. (canceled)
 78. A method for inhibiting amyloid formation, oramyloid aggregate formation, in a subject, comprising administering tothe subject a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein: L¹ is a bond; L²is a bond, —CH₂—, —(C═O)—, —(C═O)O—, or —NR²(C═O)—; G¹ is —H or R³; G²is —H, —N(R²)₂, —N(R²)(R³) or R³; R is —H or C₁₋₆ alkyl; R¹ is —F, —OH,or —O(C₁₋₆ alkyl); R² is independently, for each occurrence, —H or C₁₋₆alkyl; R³ is aryl substituted with n instances of R⁴; R⁴ isindependently, for each occurrence, —F, —OH or —OCH₃; n is, for eachoccurrence, 0, 1, 2, 3, or 4; and m is 0, 1, 2, 3, or
 4. 79. The methodof claim 78, wherein: L¹ is a bond; L² is a bond, —CH₂—, —(C═O)—,—(C═O)O—, or —NR²(C═O)—; G¹ is —H or R³; G² is —N(R²)(R³) or R³; R is Hor —CH₃; R¹ is —OH or —OCH₃; R² is —H or —CH₃; R³ is phenyl substitutedwith m instances of R⁴; R⁴ is independently, for each occurrence, —F,—OH or —OCH₃; n is, for each occurrence, 0, 1, 2, 3, or 4; and m is 0,1, 2, 3, or
 4. 80. The method of claim 78, wherein Formula (II) is ofFormula (II-a) or Formula (II-b):


81. The method of claim 80, wherein Formula (II-a) is of Formula(II-a-1) or Formula (II-a-2):

and wherein Formula (II-b) is of Formula (II-b-1) or Formula (II-b-2):


82. The method of claim 78, wherein the sum of all instances of m and nis 3-9.
 83. The method of claim 78, wherein each R³ is independentlyselected from:


84. The method of claim 78, selected from:

and pharmaceutically acceptable salts thereof.
 85. The method of claim78, wherein the subject is at risk of developing, or has been diagnosedwith, an amyloid disorder.
 86. The method of claim 85, wherein theamyloid disorder is diagnosed by detecting the presence or level ofintestinal bacterial amyloid aggregates.
 87. The method of claim 86,wherein the aggregates comprise a bacterial CsgA protein.
 88. The methodof claim 85, wherein the amyloid disorder is a neurological disorder.89. The method of claim 88, wherein the neurological disorder isParkinson's disease (PD), Lewy body dementia, multiple system atrophy,α-synucleinopathy, PD-associated constipation, PD-associated hyposmia,Huntington's Disease, Alexander's Disease, amyotrophic lateral sclerosis(ALS), or Alzheimer's Disease.
 90. The method of claim 85, wherein theamyloid disorder is intestinal dysbiosis, intestinal hyperpermeability,irritable bowel syndrome (IBS), inflammatory bowel disease (IBD),ulcerative colitis or Crohn's disease.
 91. The method of claim 78,wherein the subject suffers from gastrointestinal symptoms including oneor more of dysphagia, reduced gut motility, gastroparesis, constipation(including chronic constipation and chronic idiopathic constipation),small intestine bacterial overgrowth (SIBO), diarrhea, abdominal painand/or cramping, bloating, flatulence, hypersalivation (sialorrhea),anorectal dysfunction, dyssynergic defecation, and nausea.
 92. Themethod of claim 78, wherein the gastrointestinal symptoms are associatedwith Parkinson's Disease or Parkinsonism.
 93. The method of claim 78,wherein the amyloid aggregates comprise one or more mammalian proteins.94. The method of claim 93, wherein the mammalian proteins are selectedfrom α-synuclein, tau, Beta amyloid from Amyloid precursor protein,Medin, Apolipoprotein AI, Atrial natriuretic factor, Beta amyloid,Cystatin, IAPP (Amylin), Beta-2 microglobulin, Transthyretin, PrP,Gelsolin, Lysozyme, Huntingtin, Keratoepithelin, Calcitonin, Prolactin,Serum amyloid A, SOD1, and Immunoglobulin light chain AL.
 95. The methodof claim 78, wherein the amyloid aggregates comprise one or morebacterial or fungal proteins.
 96. The method of claim 95, wherein thebacterial protein is CsgA.
 97. The method of claim 78, wherein theamyloid aggregates are present within the gastrointestinal tract,cranial sinus, or nasal cavity of the subject.